Compositions and methods for treatment of neoplastic disease

ABSTRACT

The present invention comprises compositions and methods for treating a tumor or neoplastic disease in a host, The methods employ conjugates comprising superantigen polypeptides or nucleic acids with other structures that preferentially bind to tumor cells and are capable of inducing apoptosis. Also provided are superantigen-glycolipid conjugates and vesicles that are loaded onto antigen presenting cells to activate both T cells and NKT cells. Cell-based vaccines comprise tumor cells engineered to express a superantigen along with glycolipids products which, when expressed, render the cells capable of eliciting an effective anti-tumor immune response in a mammal into which these cells are introduced. Included among these compositions are tumor cells, hybrid cells of tumor cells and accessory cells, preferably dendritic cells. Also provided are T cells and NKT cells activated by the above compositions that can be administered for adoptive immunotherapy.

CROSS-REFERENCE TO RELATED DOCUMENTS

The Instant application is a continuation application of U.S.application Ser. No. 09/650,884 filed on Aug. 30, 2000 which claimspriority to provisional applicaton 60/151,470 filed on Aug. 30, 1999.Both of the above referenced applications are incorporated herein intheir entirety by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to immunotherapeutic compositions andmethods for treating tumors and cancer. The methods are based on theexpression of superantigen (“SAg”) alone or in combination with othermolecules in transfected host cells (tumor cells, accessory cells orlymphocytes). Other therapeutic methods are based on administering Tcells which are activated by cells engineered to express SAg and otherimmunostimulatory molecules and structures.

2. Description of the Background Art

Therapy of the neoplastic diseases has largely involved the use ofchemotherapeutic agents, radiation, and surgery. However, results withthese measures, while beneficial in some tumors, has had only marginaleffects in many patients and little or no effect in many others, whiledemonstrating unacceptable toxicity. Hence, there has been a quest fornewer modalities to treat neoplastic diseases.

In 1980, tumoricidal effects were demonstrated in four of five patientswith advanced breast cancer treated with autologous plasma that had beenperfused over columns in which Staphylococcal Protein A was chemicallyattached to a solid surface (Terman et al., New Eng. J. Med., 305:1195(1981)). While the initial observations of tumor killing effects withthe immobilized Protein A perfusion system have been confirmed, somehave obtained inconsistent results.

The explanation of these inconsistencies appears to be as follows.First, commercial Protein A is an impure preparation, as evident frompolyacrylamide gel electrophoresis and radioimmunoassays that detectedStaphylococcal enterotoxins in the preparations. Second, various methodsof immobilizing Protein A to solid supports have been used, sometimesresulting in loss of biological activity of the perfusion system. Third,the plasma used for perfusion over immobilized Protein A has often beenstored and treated in different ways, also resulting in occasionalinactivation of the system. Moreover, the substance(s) or factorsresponsible for the anti-tumor effect of this extremely complexperfusion system have not been previously defined. The system containedan enormous number of biologically active materials, including theProtein A itself, Staphylococcal proteases, nucleases, exotoxins,enterotoxins and leukocidin, as well as the solid support and coatingmaterials. In addition, several anaphylatoxins were generated in plasmaafter contact with immobilized Protein A. Finally, it was speculatedthat the biological activity of the system was due to the removal fromthe plasma by the Protein A of immunosuppressive immune complexes thatotherwise inhibit the patient's antitumor immune response.

The Staphylococcal enterotoxins that contaminate the Protein A columnsare a family of extracellular products of Staphylococcal aureus thatbelong to a well recognized group of proteins that have common physicaland chemical properties. The enterotoxins produce a number ofcharacteristic effects in humans and animals, such as emesis,hypotension, fever, chills, and shock in primates and enhancement ofgram negative endotoxic lethality in rabbits. At least some of theseeffects are due to the ability of these proteins to act as extremelypotent T cell mitogens.

Staphylococcal enterotoxins are representative of a family of moleculesknown as SAgs which are the most powerful T cell mitogens known. Theyare capable of activating 5 to 30% or the total T cell populationcompared to 0.01% for conventional antigens. Moreover, the enterotoxinselicit strong polyclonal proliferation at concentrations 10³-fold lowerthan conventional T cell mitogens. The most potent enterotoxin,Staphylococcal enterotoxin A (SEA), has been shown to stimulate DNAsynthesis in human T cells at concentrations of as low as 10⁻¹³ to10⁻¹⁶M. Enterotoxin-activated T cells produce a variety of cytokines,including IFN, various interleukins and TNF. Enterotoxins stimulateseveral other cell populations involved in innate and adaptive immunitywhich also play a major role in anti-tumor immunity, For example,enterotoxins engage the variable region of the TCR chain on exposed faceof the pleated sheet and the sides of the MHC class II molecule.

The SAg is capable of augmenting the TH-1 cytokine response by CD4+cells r while also activating NKT and NK cells. NK cell cytotoxicity isaugmented by IFN produced by SAg activated T cells. NKT cells are knownto be activated by SAgs, peptides, -galactosylceramides andlipoarabinomannans presented on CD1 receptors. Evidence points to aninvariant lectin like recognition unit on the NKT cell chain as aspecific ligand for galactosylceramide determinants on tumor cells. SAgsinduce tumor killing in vivo when given alone or conjugated to tumorassociated antibodies. They are also effective when employed ex vivo toproduce tumor sensitized T cells for the adoptive therapy of MCA 205/207tumors. SAg transfected tumor cells have shown a capacity to reducemetastatic disease in a murine mammary carcinoma model.

In addition to these common biological activities, the Staphylococcalenterotoxins share common physicochemical properties. They are heatstable, trypsin resistant, and soluble in water and salt solutions.Furthermore, the Staphylococcal enterotoxins have similar sedimentationcoefficients, diffusion constants, partial specific volumes, isoelectricpoints, and extinction coefficients. The Staphylococcal enterotoxinshave been divided into five serological types designated SEA,Staphylococcal enterotoxin B (SEB), Staphylococcal enterotoxin C (SEC),Staphylococcal enterotoxin D (SED), and Staphylococcal enterotoxin E(SEE), which exhibit striking structural similarities. The enterotoxinsare composed of a single polypeptide chain of about 30 kilodaltons (kD).All staphylococcal enterotoxins have a characteristic disulfide loopnear the middle of the molecule. SEA is a flat monomer consisting or 233amino acid residues divided into two domains. Domain I comprisesresidues 31-116 and domain II of residues 117-233 together with theamino tail 1-30. In addition, the biologically active regions of theproteins are conserved and show a high degree of homology. One region ofstriking amino acid sequence homology between SEA, SEB, SEC, SED, andSEE is located immediately downstream (toward the carboxy terminus) fromthe cysteine located at residue 106 in SEA. This region is thought to beresponsible for T cell activation. A second homologous region thatbegins at residue 147 and extends downstream is highly conserved. Thisregion is believed to mediate emetic activity. The region related toemetic activity can be omitted from enterotoxins used as therapeutics.

A sequence analysis of the Staphylococcal enterotoxins with other toxinshas revealed SEA, SEB, SEC, SED, Staphylococcal toxic shock-associatedtoxin (TSST-1 also known as SEF), and the Streptococcal exotoxins shareconsiderable nucleic acid and amino acid sequence homology. Theenterotoxins belong to a common generic group of proteins thought to beevolutionarily related.

Enterotoxins bind to MHC Class II molecules and the T cell receptor(“TCR”) in a manner quite distinct from conventional antigens.Enterotoxins engage the variable region of the TCR □ chain on an exposedface of the □ pleated sheet and the sides of the MHC Class II molecule,rather than engaging the groove of the Class II molecule likeconventional antigens. In contrast to SEB and the SEC, which have onlythe capacity to bind to the MHC class II α chain, SEA, as well as SEEand SED, in a zinc dependent manner, also interacts with the MHC classII □ chain. T cell recognition is based on the presence of the □ chainand is therefore independent of other TCR components and diversityelements. Single amino acid positions and regions important for SAg-TCRinteractions have been defined. These residues are located in thevicinity of the shallow cavity formed between the two domains. Thealanine substitution of amino acid residue Asn23 in SEB has demonstratedthe importance of this residue in SEB/TCR interaction. This particularresidue is conserved among all of the Staphylococcal enterotoxins andmay constitute a common anchor position for enterotoxin interaction withTCR V□ chains. Amino acid residues in positions 60-64 have also beenshown to contribute to the TCR interaction as do the cysteine residuesforming the intermolecular disulfide bridge of SEA. For SEC2 and SEC3,the key points of interaction in the V□ chain are located in the CDR1,CDR2 and HRV4 TCR V□-3 chain. Hence, multiple and highly variable partsof the V□ chain contribute to the formation of the enterotoxin bindingsite on the TCR. Thus far, a single and linear consensus motif in theTCR V□ displaying a high affinity interaction with particularenterotoxins has not been identified. A significant contribution of theTCR α chain in enterotoxin-TCR recognition is acknowledged as well asMHC class II isotypes. This distinctive binding mechanism ofenterotoxins which bypasses the highly variable parts of the MHC classII and TCR molecules allows them to activate a high frequency or T cellswith massive lymphoproliferation, cytokine induction and cytotoxic Tcell generation. These properties are shared by other proteins made byinfectious agents. Together, these proteins form a well recognized groupknown as SAgs.

There are two general classes of SAgs. The first includes minorlymphocyte stimulating (MLS) antigens. The second class of SAgs includesmycoplasmal, viral, and bacterial proteins such as the Staphylococcalenterotoxins. Streptococcal exotoxins. All SAgs have the followingproperties. T cell activation does not require antigen processing. Thereis no MHC restriction of responding T cells. SAgs bind to and evokeresponses from all T cells expressing V receptors, without requiringother TCR or diversity elements. CD4-CD8-α/□ T cells and γ/δ T cells arealso capable of responding to SAgs. The SAgs induce a biochemicallydistinct T cell activation pathway. Thus, SAgs interact with andactivate a much larger proportion of T cells than conventional antigens,causing massive lymphoproliferation, cytotoxic T cell generation, andcytokine secretion. A given SAg can activate up to 30% of resting Tcells compared to 0.01% for conventional antigens. As highlyrepresentative members of this family of SAgs, the enterotoxins sharethese characteristics.

The present invention features the use of SAgs in association withmolecules to produce tumor killing effects. The SAgs are useful inpeptide form and may combine with another peptide or nucleic acid toform a conjugate. The effect of the combined molecules is synergistic.These conjugates are useful when administered as a preventative ortherapeutic antitumor vaccine in tumor bearing patients. Alternatively,they may be used ex vivo to load an antigen presenting cell as a meansof immunizing a T or NKT cell population for use in adoptive therapy ofcancer. Examples of such conjugates are complexes between: SAg andglycosylceramide; SAg and apolipoproteins (Lp(a)), SAg and oxyLDL, SAgand verotoxins, SAg and GPI-ceramide (with phytosphingosine backbone),SAg and lipopolysaccharide (LPS), SAg and peptidoglycan, SAg and mannanproteoglycan, SAg and muramic acid, SAg and tumor peptides. Alsointended are SAg and Gal conjugates and glycosylated SAgs.

The present invention features the use of SAg in association orconjugated to oxidized low density lipoproteins (oxyLDL) andapolipoproteins (e.g., lipoprotein (a) (Lp(a)). OxyLDL and itsbyproducts bind to receptors on sinusoidal endothelial cells in thetumor microcirculation where they induce apoptosis, increase levels oftissue factor and activated thrombin, upregulate achesion molecules andproduce a prothrombotic state. Lp(a) is densely deposited in tumormicrocirculation and as a competitive inhibitor of plasminogen isprothrombotic. Hence, both apolipoproteins and oxyLDL not only home toreceptors on the tumor microcirculation but they also induce endothelialcell or macrophage apoptosis as well as a prothrombotic state. Theselocal effects are amplified by the presence of the conjugatedsuperantigen which induce a localized T cell immune and inflammatoryresponse collectively resulting in a potent anti-tumor response.

The present invention also features the use of the SAg in association orconjugated to verotoxins. The latter molecules have the capacity to bindto galactosylceramide receptors on tumor cells and induce apoptosis.Hence, the tumor targeting and apoptosis inducing functions of theverotoxin are coupled with the T cell immune and inflammatory responseinduced by the SAgs to produce a potent and well localized anti-tumorresponse.

The present invention features the use of SAgs in association orconjugated to mono or digalactosylceramides. The latter have beenisolated from marine sponge Aegelus mauritanius and is expressed incertain bacteria such as Sphingomonas paucimobilis. They have been shownto activate NKT cells and to induce anti-tumor effects in vivo againstseveral types of tumors. The activation of NKT cells in the presence ofthe mono and digalactosylceramides appears to be IL-12 dependent. Thebiological activity of the -galactosylceramides is observed in both monoand digalactosylceramide forms and is dependent upon the presence of ananomeric configuration on the terminal galactose. The lengths of thesphingosine base and fatty acyl chains of 23 and 15 respectively alsoappear to be optimal for production of the anti-tumor effects.

SAgs are known to be the most powerful T cell mitogens known and havebeen shown to produce anti-tumor effects in several animal models. The-galactosylceramides are known to be potent inducers of NKT cellactivation which have been shown to produce an anti-tumor effect in anIL-12 dependent manner. In the present invention SAgs are combined with-galactosylceramides biochemically as conjugates and genetically withina cell which expresses the newly synthesized protein-boundgalactosylceramide on the cell surface. The newly synthesized conjugatesin native form or expressed in or on the cell produce a synergisticanti-tumor effect due to the activation of T cells and NKT cellpopulations.

Furthermore, in the present invention the SAg--galactosylceramides areexpressed in tumor cells, dendritic cells (“DC”) or a hybrid cell madeby fusing a tumor cell and a DC. The use of DCs or DC/tumor cell hybrids(DC/tc) to present the SAg-galactosylceramides fusion constructs orconjugates provides the optimal costimulation for activation of a tumorspecific T cell population. The use of a tumor cell or a DC/tc providesin addition to costimulation, expression of the tumor antigen itself toactivate anti-tumor T and NKT cell clones which are tumor specific.Hence, an optimal cell is a DC/tc which expresses SAg and SAg-anomericgalactosylceramides.

The SAg--galactosylceramide conjugates are useful in the presentinvention. However, there are distinct differences and advantages toproducing and expressing the SAg-galactosylceramide conjugates within acell. First, final products are quite different. One involves theenterotoxin--galactosylceramide in free form whereas the other involvescell associatedenterotoxin-galactosylceramide which includes enterotoxinnucleic acids and peptides. In the cell both enterotoxins and-galactosylceramides are associated with numerous intracellular andmembrane structures such as MHC, costimulatory and adhesion molecules,heat shock proteins, membrane glycolipids and glycosphingolipids. whichmay improve immunogenicity and antigen presentation. They may also betransported in various vesicles and exosomes which may provideadditional immunogenicity. With the addition of appropriate signalssequences and association with molecules involved in the antigenpresenting pathways such as the invariant chain, TAP and LAMP moleculesthe conjugates may be routed in the cell to the MHC class I, class II orCD1 receptor. Therefore, enterotoxin and -galactosylceramides producedwithin a cell is presented to the host's immune system in an entirelydifferent form compared to the purified enterotoxin polypeptide.

Unlike free enterotoxin polypeptide or -galactosylceramide, SAgtransfected tumor cells, DCs or DC/tc present enterotoxins to the T cellsystem in association and or conjugated to tumor associated antigensincluding mutated normal structures or fusion structures, costimulatoryand adhesion molecules. Indeed, the coadministration of SAg with tumorantigen would be expected to produce a heightened response to the tumorantigens while preventing the clonal deletion which occurs with SAgalone. Liu et al., Proc. Natl. Acad. Sci., 88: 8705-8709, (1991);McCormack et al., Proc. Natl. Acad. Sci., 91: 2086-2090, (1994); Coppolaet al., Int. Immunol., 9: 1393-403, (1997). Hence, the coadministrationof SAg-galactosylceramide and tumor associated antigens would induce apredictably heightened tumor specific response by the host. Thisprediction was borne out by the Applicant's work showing that SAgtransfection of tumor cells abolished the tumorigenicity of 4T1 mammarycarcinoma cells, significantly reduced the number of establishedmetastases and prolonged survival compared to untreated controls.(Pulaski, Terman, et al., American Association of Cancer Research, April1999 and submitted to Proc. Natl. Acad. Sci, 1999).

SAg transfected tumor cells in vivo are effective in an additionalmanner which does not apply to SAg polypeptide. Ingestion of apoptoticcells by DCs augments the immunogenicity of tumor cells. Fields et al.,Proc. Natl. Acad. Sci., 95: 9882-9887, (1998); Albert et al., Nature,392: 86-89, (1998). DCs are acknowledged as the premier accessory cellfor antigen presentation. They have been shown to ingest apoptotic cellsand nucleic acids and process them for presentation to host T cells inthe context of costimulation, adhesion and MHC molecules. Akbari et al.,J. Exp. Med., 189: 169-177, (1999). Therefore, following apoptosis ofSAg transfected tumor cells and ingestion by DCs, SAg-encoding nucleicacid as well as tumor associated nucleic acids in the transfected cellswould produce additional anti-tumor responses. Purified polypeptideenterotoxins do not share with the SAg transfectants this property ofenhanced immunogenicity following ingestion and processing by DCs.

There are enormous structural and functional differences between thepolypeptide enterotoxin and SAg-transfected tumor cells. The startingmaterials are different i.e. peptides vs nucleic acids and the productis different i.e. polypeptide vs enterotoxin transfected cell in whichthe SAg is may exist in nucleic acid and peptide form associated with avast number of intracellular and membrane structures. Some of thesestructures may actually improve the T cell activating function of SAgssuch as deoxyribonucleic acids, ribonucleic acids, tumor associatedantigens, heat shock proteins, costimulatory molecules and adhesionmolecules and endosomes. Cellular SAg peptides or nucleotides exist inassociation with tumor associated antigens, costimulants, adhesionmolecules, heat shock proteins and MHC molecules, GPI-ceramides or SAgreceptors (digalactosylceramides) which improve the immunogenicity ofthe tumor antigens. Therefore, these structural and functionaldifferences between the polypeptide SAg and the enterotoxin transfectedtumor cells clearly show that SAg transfected tumor cells have a fargreater potential than the polypeptide to induce a tumor specificresponse.

Moreover, SAg transfected tumor cells possess an additional uniqueproperty not shared by the polypeptide SAg. SAg-transfected tumor cellsdisplay the metastatic phenotype of the tumor cells which enables themto colonize and traffic to metastatic sites in vivo. Once localized tomicrometastatic sites the transfectants expressing SAg induce a potenttumor specific T cell response. In contrast, the purified polypeptideSAg unassociated with a tumor cell would have no capacity whatsoever tocolonize metastatic sites.

The present invention also provides SAg-encoding nucleic acid,preferably DNA, fused with (or cotransfected with ) a nucleic acidencoding another molecule. The transfected cells include tumor cells,accessory cells e.g., DCs, tumor cell/accessory cell (e.g., DC) hybrids.The expression of molecules in addition to enterotoxins by these cellsserves the following functions:

1) enhance the immunogenicity of the SAg transfected cell by providingnucleic acids encoding an additional potent immunogen. Examples wouldinclude tumor associated antigens or mutated normal antigen or fusionpeptides in tumor cells, an immunogenic bacterial product such asStaphylococcal adhesin protein A, LPS, □-glucans, and peptidoglycans,costimulatory and adhesion molecules, heat shock protein, growth factorreceptors such as Her/neu and tumor markers such as PSA.

2) assist in tumor killing activity by the SAg transfected cell whenlocalized to tumor sites. by providing nucleic acids encoding thefollowing: angiogenesis antagonists, chemoattractants such as C5a,chemokines such as RANTES, hyaluronidase and coagulase and CD44isoforms.

3) increase the binding of immunogenic substances to the surface of theSAg transfected cell by providing nucleic acids encoding the following:CD1 receptors, CD14 receptors, SAg receptors

4) increase the production of SAg in the SAg transfected cell byproviding nucleic acids encoding the following: cell cycle proteins,amplified oncogenes, and signal transduction molecules.

5) assist in trafficking of SAg to class I or class II pathway in theSAg transfected cell by providing nucleic acid encoding the following:the invariant chain, the LAMP1 proteins and TAP proteins.

6) induction of a local tumoricidal response by intratumoral injectionof nucleic acids encoding the following: oxyLDL receptor and SAgreceptor, chemoattractants, chemokines.

SUMMARY OF THE INVENTION

The present invention comprises a method for treating cancer in a hostcomprising providing conjugates, fusion proteins or naked nucleic acidsof superantigen and additonal molecule(s) which produce an tumoricidalresponse. The addtional molecule serves the following functions: 1) totarget a receptor (digalactosylceramide) expressed on tumor cells invivo and induce tumor cell apoptosis e.g., SAg-verotoxin conjugates. 2)to target receptors expressed on tumor sinusoidal endothelium, induceapoptosis and a prothrombotic state e.g. SAg-oxyLDL conjugates andSAg-Lp(a) conjugates 3) to activate a dormant population of tumoricidalNKT cells e.g. SAg-digalactosylceramides, SAg-GPI-digalactosylceramide(phytosphingosine) complexes. 4) target receptors for integrinsexpressed on tumor microvasculature e.g., SAg-RGD conjugates. 5) nakedDNA administered intratumorally induces tumor cell expresson in vivo ofreceptors for ligands which produce apoptosis and inflammation e.g,naked DNA SAg-oxyLDL receptor, SAg-LOX-1 receptor, SAg-SREC receptor..

Sickled erythrocytes are useful in the present invention since they havenatural ligands for integrins expressed on tumor neovasculature whichfacilitates their targeting to the tumor endothelium. Sicklederythrocyte membranes acquire oxyLDL using fusigenic techniques withoxyLDL containing liposomes and apoproteins via gene transfection in thenucleated pre-reticulocyte phase. The oxyLDL and apoproteins expressedby the sickled cells facilitates targeting to oxyLDL, LOX-1 and SRECreceptors present on the tumor microvasculature. These erythrocytes arealso useful for carrying nucleic acids for transfection of the tumorendothelial cells in vivo. Vesicles derived from sickled erythrocytesare more rigid, prothrombotic and target the tumor microvascularturemore effectively than the parent cell. They also carry oxyLDL toreceptors on tumor endothelium. Likewise, vesicles, exosomes orSAg-GPI-digalctosylceramides shed from from SAg transfected tumor cellsare capable of inducing potent tumoricidal responses and are useful inthe present invention.

In addition, bacterial expression systems are useful for the expressionof SAg in association with other anti-tumor molecules. The yeast secmutant is used to produce a SAg-ceramide conjugate in which thesphingosine portion of the ceramide is a phytosphingosine. Sphingomonaspaucimobilis which naturally expresses α-galactosylceramide istransfected with SAg nucleic acids which results in the shedding ofSAg-α-galactosylceramide complexes.

The present invention comprises a method for treating cancer in a hostcomprising providing cells transfected with a gene that express and/orsecretes a SAg or T cells activated by the transfected cells to thehost. The cells are transfected in vivo or in vitro. SAgs may activate Tcells or NKT cells in the host. These same transfectants may be used tostimulate a population of T cells or NKT cells ex vivo which areprovided to the host as tumor specific effector cells in adoptiveimmunotherapy. The transfected cells may be, for example, tumor cellsaccessory cells, DCs muscle cells, immunocytes, fibroblasts. Whentransfected in vitro the cells can be xenogeneic to the host, from thesame species as the host or host cells.

For in vivo immunization, tumor cells are transfected with nucleic acidsencoding SAgs together with a carbohydrate modifying enzyme such asgalactosyl transferase to produce the Gal epitope, Staphylococcalhyaluronidase, Streptococcal capsular polysaccharide, Staphylococcalerythrogenic toxin, Staphylococcal Protein A, Staphylococcal □hemolysin, Staphylococcal coagulase, costimulants such as B7-1 and B7.2,chemoattractants and chemokines. SAgs are also cotransfected into tumorcells with gene clusters encoding the biosynthesis of highly immunogenicmicrobial Lipid A, membrane or capsular polysaccharides, lipoproteinsand peptidoglycans. Nucleic acids are useful when transfected alone.However combinations are preferred. The cotransfection into tumor cellsof the SAg-encoding nucleic acid together with the nucleic acidsencoding Gal or GalCer biosynthesis is particularly useful. Thecotransfection into tumor cells of the nucleic acid encoding SAg withnucleic acids encoding Staphylococcal erythrogenic toxins andhyaluronidase allows the transfected tumor cells to simulate the in vivoinflammatory activity of a Staphylococcus or leukocyte or macrophage bysecreting enzymes and toxins which induce a sterile cellulitis in tumorsites.

Further provided are tumor cells transfected with nucleic acid encodingstructures such as the erb/Neu gene which upon administration to thehost promotes tumor cell trafficking and colonization of micrometastaticsites. Amplified oncogenes linked to SAg nucleic acids provide the locusand energy for expression or overexpression of both gene products. Thus,provided herein are tumor cells transfected with SAg-encoding nucleicacid together with nucleic acid encoding other oncogenes, amplifiedoncogenes and transcription factors, angiogenic factors such asangiostatin, angiogenesis receptors such as VEGF, tumor growth factors,tumor suppressors, cell cycle proteins and key proteins engaged in theantigen routing and processing pathway. In one example, the microbialSAg and erb/Neu nucleic acids are cotransfected into tumor cells. Thesenucleic acids may also linked to an inducible gene such as that encodingmetallothionein or corticosteroid receptors. In this way, the cells areactivated by exogenous delivery of corticosteroids or a heavy metal onlyafter a suitable period of time has lapsed to allow them to localize inmetastatic sites in vivo

Tumor cell transfectants are also useful ex vivo to immunize a T cell orNKT cell population producing tumor specific effector cell populationfor adoptive immunotherapy of cancer. These immunizing tumor cells aretransfected with nucleic acids encoding SAgs and the SAg receptor. Thelatter transfectants are capable of binding exogenous SAg forpresentation to a T cell population. In addition, tumor cells aretransfected with nucleic acids encoding CD1 receptors which are capableof binding exogenous glycosylceramides and lipoarabinans free or boundto SAgs for presentations to T or NKT cells. Similarly, tumor cells aretransfected with nucleic acids encoding the CD14 receptor which bindexogenous peptidoglycans and LPS's, free or bound to SAgs forpresentation to T cells.

Likewise, the nucleic acids encoding the mannose receptor aretransfected into tumor cells which are capable of binding a broad rangeof glycosylated SAgs for presentation to T cells. The present inventionprovides detailed methods for preparation of the SAg-glycosylceramide,SAg-LPS, SAg-peptidoglycan complexes as well as glycosylated SAgs whichare loaded onto their respective receptors expressed on tumor cells,accessory cells and, in some instances, immunocytes. For ex vivo use,any prokaryotic or eukaryotic cell may be used which is transfectablewith nucleic acid encoding SAgs to provide surface expression of the SAgor constructs expressed on tumor, accessory cell or immunocytetransfectants. When the transfected cells are not host tumor cells, thecells additionally express a tumor associated antigen expected to bepresent on the host's cancer cells.

Also provided is a tumor specific T cell or NKT cell population which isactivated by SAgs or the tumor cell transfectants above to produce apopulation of tumor specific effector cells useful in adoptiveimmunotherapy. After ex vivo stimulation, the T cells or NKT cells usedfor adoptive immunotherapy should preferentially express CD44 whichindicates that they are capable of trafficking and homing to tumorsites. Additionally, the T cell population used for ex vivo immunizationis engineered to overexpress the TCR variable V□ and invariant Vα sitesspecific for SAg and glycosylceramide binding respectively and toproduce IFN by exogenous delivery of corticosteroids or a heavy metal. Aparticularly useful population of therapeutic tumor specific effector Tcells or NKT cells which demonstrates overexpressed CD44 together withV□ variable and Vα invariant regions and high IFN production. Alsoprovided are methods for reactivating anergic T cells in cancer patientsby transfecting nucleic acids encoding the SAg receptors to produce a Tcell population which may now be stimulated with exogenous SAgs.

Compositions which mimic SAgs are used in place of native SAgs for invivo administration in order to circumvent the problem of naturallyoccurring SAg-specific antibodies. The SAg mimics are largely comprisedof nucleotides or oligonucleotide-peptide chimeric constructs which arespecific for tumor cells expressing SAg receptors (via the nucleotide)while retaining their SAg specificity for the TCR (via the peptide). Theclass II binding site of the SAg may optionally be eliminated or mutatedto minimize SAg peptide binding to MHC class II receptors in vivo. Themolecule may be composed entirely of nucleotides for which there are nonaturally occurring antibodies. In addition, carriers are provided forin vivo transfection of tumors by nucleic acids encoding SAgs or othernucleic acid constructs given in Table I. Phage displayed tumorneovasculature ligands may also carry nucleic acids encoding SAgs orother constructs.

The constructs and method are used to treat any solid tumor such ascarcinoma, melanoma and sarcoma or cancer of hemopoietic origin, such aslymphomas and leukemias which may or may not form solid tumors.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art of this invention. Although methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the present invention, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting. Otherfeatures and advantages of the invention will be apparent from thefollowing detailed description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic diagram of the cloning of the SEB gene into thepHβ=Apr1-neo vector. The coding region of the SEB gene was amplifiedwith PCR primers. The upstream primer (SEB 1) has a SalI site at its5′end and the downstream primer (SEB2), a BamHI site. Both the pHbApr1-neo vector and the amplified SEB insert were digested with SalI andBamHI, ligated and transformed into XL1-Blue competent cells. The finalconstruct was verified by restriction enzyme and sequence analyses.

FIG. 2. Cloning of the SEB gene into the

neo vector. Clones 1-5 contained the SEB insert (coding region 801 bp)and the

neo vector (10 kb). All DNA was digested with SalI and BamHI andelectrophoresed on a 1% agarose gel in 1×TAE buffer.

FIG. 3. Alignment of the published SEB coding sequence (SEQ ID NO:1) andthe newly constructed SEB gene (SEQ ID NO:2) in

neo vector (Clone #2). Clone #2 was sequenced with 4 primers: SEB1, 2,3, and 4. SEB1 and 2 (SEQ ID NOS:3-4) are the PCR primers that were usedfor the amplification of the SEB gene. SEB 3 (TATGAAAGTTTTGTATGATGAT)(SEQ ID NO:5) and SEB 4 (SEQ ID NO:6) (AGTGACGAGTTAGGTAATCT) areinternal primers. The final sequence was confirmed by the multipleoverlapping of sequences and aligned with the published SEB sequence. Itis a perfect match. The start codon (ATG) and the stop codon (TGA) areunderlined. The upstream and the downstream sequences are the human□-actin promoter and the SV40 polyA sequences in the pHβ^(a)-Apr1 neovector with the addition of SalI and BamHI restriction enzyme sites.TABLE I Therapeutic Constructs And Preferred Conditions Of Use I. CELLS:Tumor Cells, DCs or DC/Tumor Cell Hybrids (DC/tc) USE: In vivo and Exvivo PURPOSE A. In Vivo Preventative or Therapeutic Vaccine (EstablishedTumor) Accomplish by transfecting or co-transfecting with nucleic acidencoding superantigen plus one or more of the following: 1.Superantigens 2. Enzyme that modifies carbohydrate to induce Gal orGalCer epitope expression 3. Functional hyaluronidase from microbial orhuman sources 4. Staphylococcal or streptococcal erythrogenic toxin 5.Staphylococcal protein a or a domain thereof 6. Staphylococcal hemolysinand functional microbial toxins 7. Functional microbial or humancoagulase 8. Costimulatory protein 9. Chemoattractants 10. Chemokines11. Nucleic acids encoding biosynthesis of lipopolysaccharides 12.Nucleic acids encoding biosynthesis of glycosylceramides 13. Nucleicacids encoding biosynthesis of microbial membrane or capsularlipoproteins and polysaccharides 14. Oncogenes, amplified oncogenes andtranscription factors 15. Angiogenic factors and receptors 16. Tumorgrowth factor receptors 17. Tumor suppressor receptors 18. Cell cycleproteins 19. Heat-shock proteins, ATPases and G proteins 20. Proteinsengaged in antigen processing, sorting and intracellular trafficking 21.Inducible nitric oxide synthase (iNOS) 22. apolipoproteins (e,g,. Lp(a))transfected into tumor cells & sickled erythrocytes used for targetingtumor microvasculature 23. LDL and oxyLDL receptors (e.g., SCEPreceptor) transfected into tumor cells and sickled erythrocytes & usedfor targeting to tumor microvasculature B. Ex Vivo Immunization of Tand/or NKT cells to Produce Tumor Specific Effector Cells (for AdoptiveImmunotherapy)* Accomplish by (i) transfecting or co-transfecting tumoror accessory cells with nucleic acid encoding the following, or (ii)providing immobilized molecules or receptors that present thefollowing: 1. Superantigen 2. Superantigen receptor and transcriptionfactor with bound superantigen 3. CD1 receptor binding and/or expressingsuperantigen-glycosyl ceramide complex 4. CD14 receptor binding orexpressing superantigen- lipopolysaccharide orsuperantigen-peptidoglycan complex 5. Mannose receptor bindingglycosylated superantigen 6. Glycophorin receptor 7. Superantigen-tumorpeptide(s) complex on MHC or CD1-bearing APC in soluble or immobilizedform C. Therapeutic Molecules or Complex Applied to Transfected orUntransfected Tumor cells or Accessory Cells; or MHC class I, class II,CD1, Superantigen receptor or CD14 receptor: 1. Superantigen (whereincell may express Gal) 2. Glycosylated superantigen 3. Superantigencomplex with a. glycosyl ceramide b. lipopolysaccharide c. peptidoglycand. mannan proteoglycan e. muramic acid f. tumor peptide g.glycosylceramides with terminal Gal(α1-4)Gal e.g. globotriosylceramideand galabiosylceramide h. Conjugates of SAg-(Gb2 or Gb3 or Gb4) i.Conjugates of SAg-(Gb2 or Gb3 or Gb4)-CD1 j. GPI anchored conjugates:SAg-GPI-(Gb2 or Gb3 or Gb4) l. GPI anchored conjugates: SAg-GPI-(Gb2 orGb3 or Gb4)- CD1 m. Conjugates of SAg polypeptide or nucleic acid withVerotoxin n. Conjugates of SAg Polypeptide or nucleic acid withVerotoxin A or B subunit o. Conjugates of SAg polypeptide or nucleicacid with IFNα receptor peptides homologous to verotoxin p. Conjugatesof SAg polypeptide or nucleic acid with CD19 peptides homologous toverotoxin q. Conjugates of SAg polypeptide or nucleic acid with Arg-Gly-Asp or Asn-Gly-Arg r. Conjugates of SAg polypeptide or nucleic acid withLDL, VLDL, HDL s. Conjugates of SAg polypeptide or nucleic acid withApolipoproteins (e.g., Lp(a), apoB-100, apoB-48, apoE) t. Conjugates ofSAg polypeptide or nucleic acid with oxyLDL, oxyLDL mimics, (e.g.,7□-hydroperoxycholesterol, 7□-hydroxycholesterol, 7-ketocholesterol,5α-6α-epoxycholesterol, 7□-hydroperoxy-choles-5-en-3b-ol,4-hydroxynonenal (4- HNE), 9-HODE, 13-HODE and cholesterol-9-HODE) u.Conjugates of SAg polypeptide or nucleic acid with oxyLDL by products(e.g. lysolecithin, lysophosphatidylcholine, malondialdehyde,4-hydroxynonenal) v. LDL & oxyLDL receptors (e.g., LDL oxyLDL,acetyl-LDL, VLDL, LRP, CD36, SREC, LOX-1, macrophage scavengerreceptors) as polypeptide or nucleic acid alone or with SAg polypeptideor nucleic acid intratumorally II. CELLS: Specialized Tumor SpecificEffector Cells (T and/or NKT Cells) USE: Adoptive Immunotherapy In VivoPURPOSE: A. CD44 Expression on T cells or NKT Accomplished by: (i)Superantigen stimulation; and/or (ii) transfection with nucleic acidencoding CD44 and/or (iii) transfection with nucleic acid encodingglycosyltransferase B. Chimeric TCR with:    Invariant a chain site forbinding GalCer and    Vβüchain site for binding superantigen C. Dual TCRV□ chains with sites for superantigen binding D. T cells or NKT cellswith overexpressed VβNregion specific for a given superantigen E. Tcells or NKT cells with lowered signal transduction threshold III.MOLECULES:   Superantigen mimics USE: In Vivo Administration A.Superantigen receptor-binding oligonucleotides B. Superantigenoligonucleotide-peptide conjugate    Oligo nucleotide is specific forsuperantigen receptor on tumor    cells    Peptide has deleted class IIbinding site and intact TCR binding    site C. Phage displayed integrinligand on tumor neovasculature - carrier for superantigen-encodingnucleic acid. IV. CARRIERS: for nucleic acid encoding superantigen USETransfection of Tumors In vivo A. Sickled erythrocytes that target tumorneovasculature B. Phage displayed tumor neovascular integrin andsuperantigen receptor carrying superantigen nucleic acids V. CARRIERS:constructed to co-express superantigen conjugates or complexes with:Glycosylceramide αGal Lipopolysaccharides Peptidoglycans USETransfection of Tumor Cells and/or DCs and/or DC/tc's - in vivo or exvivo. A. Liposomes B. Proteosomes

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides methods and materials for treating cancerrelated to the polypepide or nucleic acid conjugates or fusionscomprising SAg with other molecules that synergize or cooperate with SAgin the induction of an anti-tumor response. The present invention alsoprovides materials and methods for treating cancer related totransfection of cells with nucleic acid that encode a SAg and/or anotherpolypeptide. The cells can be transfected in vivo or in vitro. Theexpression of the SAg polypeptide activates host immunocytes, such as Tor NKT cells.

As used in this application, T cells are defined as any class oflymphocytes that undergo maturation and differentiation in the thymus.They include, but are not limited to NK cells, NKT cells and/T cells andmay be known as cytotoxic, helper or suppressor T cells or they may bedefined by the expression or type of CD or TCR present. The sametransfected nucleic acid molecule, or a separate nucleic acid molecule,can also encode another polypeptide such as an adhesion molecule,glycosyltransferase, glycosidase, CD44, cytokine, tumor associatedantigen, costimulatory molecule, and the like. In addition, cellstransfected in vitro or ex vivo with any of these nucleic acids as wellas T cells activated by these transfected cells are administereddirectly to a cancer-bearing host. Cells transfected in vitro or ex vivoas well as cells activated ex vivo may additionally express a tumorassociated antigen expected to be present on host cancer cells. Further,cells transfected with nucleic acid that encodes a SAg polypeptide isalso be used as a vaccine to immunize a host against a cancer previouslypresent in the host or a cancer that is likely to develop in the host.For example, a host can be vaccinated against a particular cancer byadministering tumor cells transfected with nucleic acid encoding a SAg.Alternatively, a SAg transfected cell is used to activate a host T cellpopulation in vitro. This activated T cell population is thenadministered to a host as a cancer treatment (immunotherapeutic agent).Once activated ex vivo or in vivo, these T cells are expanded withcytokine treatment such as IL-2 treatment.

Cells to be “transfected” include accessory cells, immunocytes,fibroblasts, or tumor cells. Accessory cells may include, withoutlimitation, endothelial cells, DCs, monocytes, macrophages as well as Band T lymphocytes which can play an “accessory” as well as directeffector role in an immune response. When transfected in vitro, thecells can be xenogeneic, allogeneic to the host to provide, among otherthings, additional immunogenicity. Preferably, the transfected cellsthat are administered to a host, preferably a human, are syngeneic orautologous (or autochthonous).

Cells transfected with nucleic acid encoding a SAg may also express atumor associated antigen that is potentially present on host cancercells. For example, nucleic acid encoding a known tumor antigen aretransfected into the SAg-containing cell, or a tumor cell thatendogenously contains many different tumor antigens are transfected withSAg-encoding nucleic acid. In the latter case, additional nucleic acidsencoding other polypeptides are transfected into the tumor cell. Forexample, nucleic acid encoding a carbohydrate modifying enzyme such asα1,3-galactosyltransferase, adhesion molecule, costimulatory moleculesuch as B7-1 and B7-2, MHC class I molecule and/or MHC class II moleculeare cotransfected into tumor cells together with nucleic acid encoding aSAg.

SAg-encoding nucleic acid can encode a mutant, variant, and/or modifiedform of a SAg. These forms can be used to transfect T cells, alone or incombination with wild-type SAg-encoding acid. In addition, tumor cellsare provided with the capacity to colonize sites of metastases and theability to locally hydrolyze surrounding tumor ground substance andneovasculature by transfection of key bacterial Staphylococcal andStreptococcal enzymes, toxins and capsular polysaccharides which conferupon the tumor cell additional tumor killing properties andimmunogenicity. The transfected genes include staphylococcalhyaluronidase (tissue spreading factor), Staphylococcal erythrogenictoxin and Streptococcal capsular polysaccharide. The tumor cell may thusbe capable of mimicking the tissue invasive and destructive propertiesof the Streptococcus and Staphylococcus as they produce a sterilecellulitis localized to tumor sites.

These methods are used to treat any solid tumor such as carcinoma,melanoma, and sarcoma, or cancers of hematopoietic origin such asleukemia and lymphomas. This invention also provides for T cells or NKTcells including γ/δT cells which after activation by SAgs in native ormutant form or transfected into tumor cells express surface phenotypeswhich enhance their ability to traffic efficiently to tumor sites invivo. Such phenotypes include CD44 and/or selective V□ expression. Inresponse to these SAg stimulants, the T cells produce TH1 cytokines and,in particular, IFNγ and IL-2.

Further, provided are methods of overcoming the T cell unresponsivenessof cancer patients by transfection of T cells from tumor bearing hostwith the nucleic acids encoding the SAg receptor thus enabling thesecells to be reactivated by exogenous SAg and used for adoptiveimmunotherapy in the same cancer patient. Provided herein are SAgoligonucleotide and oligonucleotide-peptide compositions capable oftargeting and delivering SAgs to tumor sites in vivo without eliminationby circulating naturally occurring SAg specific antibodies prevalent inthe human cancer patients. Provided also are compositions and methodsfor delivery of therapeutic nucleic acid constructs to tumor sites invivo using therapeutic genes carried by erythrocytes from patients withsickle cell anemia which have the unique capability of adhering to siteson tumor neovasculature.

1. Cancer

This invention is used to treat any type of cancer in a host at anystage of the disease. More particularly, the cancer is a solid tumorsuch as a carcinoma, melanoma, or sarcoma. This invention is used totreat cancers of hemopoietic origin such as leukemia or lymphoma, thatinvolve solid tumors. A host is any animal that develops cancer and hasan immune system such as mammals. Thus, humans are considered hostswithin the scope of the invention. Since the invention providesSAg-transfected cells as a vaccine, a cancer is one that a host islikely to develop based on family history or other criteria. In thiscase, the host is one that is susceptible to cancer.

2. Nucleic Acid

The term nucleic acid as used herein encompasses both RNA and DNA,including cDNA, genomic DNA, and synthetic (e.g., chemicallysynthesized) DNA. The nucleic acid can be double-stranded orsingle-stranded. Where single-stranded, the nucleic acid can be thesense strand or the antisense strand. The term isolated nucleic acidmeans that the nucleic acid is not immediately contiguous with both ofthe sequences with which it is immediately contiguous (one on the 5′ endand one on the 3′ end) in the naturally occurring genome of the organismfrom which it is derived. For example, an isolated nucleic acid moleculecan be, without limitation, a recombinant DNA molecule of any length,provided nucleic acid sequences normally found immediately flanking thatrecombinant DNA molecule in a naturally occurring genome are removed orabsent. Thus, an isolated nucleic acid molecule includes, withoutlimitation, a recombinant DNA that exists as a separate molecule (e.g.,a cDNA or a genomic DNA fragment produced by PCR or restrictionendonuclease treatment) independent of other sequences as well asrecombinant DNA that is incorporated into a vector, an autonomouslyreplicating plasmid, a virus (e.g., a retrovirus, adenovirus, or herpesvirus), or into the genomic DNA of a prokaryote or eukaryote. Inaddition, an isolated nucleic acid can include a recombinant DNAmolecule that is part of a hybrid or fusion nucleic acid sequence.

Typically, regulatory elements are nucleic acid sequences that regulatethe expression of other nucleic acid sequences at the level oftranscription and/or translation. Thus, regulatory elements include,without limitation, promoters, operators, enhancers, ribosome bindingsites, transcription termination sequences (i.e., a polyadenylationsignal), and the like. In addition, regulatory elements can be, withoutlimitation, synthetic DNA, genomic DNA, intron DNA, exon DNA, andnaturally-occurring DNA as well as non-naturally-occurring DNA. It isnoted that isolated nucleic acid molecules containing a regulatoryelement are not required to be DNA even though regulatory elements aretypically DNA sequences. For example, nucleic acid molecules other thanDNA, such as RNA or RNA/DNA hybrids, that produce or contain a DNAregulatory element are considered regulatory elements. Thus, recombinantretroviruses having an RNA sequence that produces a regulatory elementupon synthesis into DNA by reverse transcriptase are isolated nucleicacid molecules containing a regulatory element even though therecombinant retrovirus does not contain any DNA.

3. Transfection

The term “transfection,” of a nucleic acid into a cell, as used hereinis intended to include “transformation,” “transduction,” “gene transfer”and the like, as they are commonly used in the art. “Transfection” isnot intended to be limited to transfer of nucleic acid into a cell bymeans of an infectious particle such as a retrovirus, as the term mayhave been used originally. Rather any form of delivery and introductionof a nucleic acid molecule, preferably DNA, into a cell, whether in theform of a plasmid, a virus, a liposome-based vector, or any othervector, so that the nucleic acid is expressed in the cell and itsprotein product(s) made, is included within the definition of“transfection.”

When a nucleic acid is said to “encode” a product other than a protein,this language is intended to mean that it encodes the necessaryproteins/enzymes that are involved in, or required for, the synthesis ofthat product. For example, if a DNA molecule is said to encode LPS, itclearly encodes one or more proteins (enzymes) that are involved in thebiosynthesis of LPS. If a nucleic acid is said to “encode thebiosynthesis” of a structure, it means that the nucleic acid encodes theenzymes that participate in the creation of that structure. Inparticular for the carbohydrate structures referred to herein, thenucleic acids used in the invention are introduced into a cell thatnormally does not make, or makes little of, the carbohydrate structureso as to provide to that cell the genetic material for an enzyme orenzymes that generate the carbohydrate structure or modify a differentcarbohydrate structure to that one indicated. As a further example, DNAencoding a tumor antigen may directly encode a protein/peptide tumorantigen, or alternatively, may encode proteins or peptides that eithercontribute structurally to, or catalyze the synthesis of, a tumorantigen which is partly protein (e.g., lipoprotein or proteoglycan) ortotally non-protein (e.g., a glycolipid). The invention provides methodsof treating cancer in a host by transfecting cells with SAg-encodingnucleic acid. Suitable host or non-host cells for transfection include,without limitation, endothelial cells, DCs, monocytes, macrophages, Bcells, T cells, immunocytes, muscle cells, fibroblasts, NK cells, NKTcells (TCR α□⁺ CD4^(neg) and CD8^(neg)), γ/δ T cells and tumor cells.The terms accessory cell and antigen presenting cell (APC) can be usedinterchangeably and include cells having the ability to process andpresent antigens to T cells as well as to provide both defined and lesswell defined growth and differentiation factors (costimulatory signals)during an ongoing immune response.

Cells are transfected in vivo or in vitro. When transfected in vivo, thecells are of host origin. When transfected in vitro, the cells areautologous, allogeneic, or xenogeneic to the host to provide additionalimmunogenicity. In addition to being transfected with nucleic acidencoding a SAg, the cells are transfected with nucleic acid encoding anyother polypeptide including, without limitation, agalactosyltransferase, staphylococcal hyaluronidase and/or erythrogenictoxin, streptococcal capsular polysaccharide, CD44, tumor antigen,costimulatory molecule such as B7-1 and B7-2, adhesion molecules, MHCclass I molecule and/or MHC class II molecule. Nucleic acids encodingthe molecules are cotransfected with the SAgs. But for others, includingbut not limited to Staphylococcal hyaluronidase, erythrogenic toxin,Streptococcal capsular polysaccharide and CD44 genes, the nucleic acidsencoding the SAgs are fused to other nucleic acids resulting inexpression of a fusion protein.

Methods for in vivo and in vitro transfection of cells are well known.For example, two books in the series Methods in Molecular Medicinepublished by Humana Press, Totowa, N.J., describe in vivo and in vitrotransfection protocols that are adaptable to the present invention(Vaccine Protocols edited by Robinson et al., (1996) in Gene TherapyProtocols edited by Robbins et al., Humana Press, Totowa, N.J. (1997)).Transfection protocols are also discussed elsewhere ((Sambrook, J. etal., Molecular Cloning, Second Edition, Cold Springs Harbor LaboratoryPress, Plainview, N.Y., (1989)). In addition, use of various vectors totarget epithelial cells, use of liposomal constructs, methods oftransferring nucleic acid directly into T cells, hematopoietic stemcells, and fibroblasts, methods of particle-mediated nucleic acidtransfer to skin cells, and methods of liposome-mediated nucleic acidtransfer to tumor cells have been described elsewhere. (Felgner, P L etal., Cationic Lipids for Intracellular Delivery of Biologically ActiveMolecules, U.S. Pat. No. 5,459,127, issued Oct. 17, 1995; Felgner, P L,Cationic Lipids for Intracellular Delivery of Biologically ActiveMolecules, U.S. Pat. No. 5,264,618, issued Nov. 23, 1993; Felgner, P L,Exogenous DNA Sequences in a Mammal, U.S. Pat. No. 5,580,859 issued Dec.3, 1996; Felgner, P L, A Protective Immune Response in a Mammal byInjecting a DNA Sequence, U.S. Pat. No. 5,589,466 issued Dec. 31, 1996).

Further, use of ligand-based nucleic acid carriers to effecttransfection of mammalian cells in vivo has been described elsewhere (Wuet al., J. Biol. Chem., 262:4429-4432 (1987); Wu et al., J. Biol. Chem.,263:14621-14624 (1988); Wu et al., J. Biol. Chem., 264:16 985-16987(1989); Wu et al., J. Biol. Chem., 266:14338-14342 (1991); and GarriguesJ et al., Am. J. Path. 142:607-622 (1993)). Briefly, plasmid DNA isconjugated to a desialylated branched carbohydrates such as orosomucoidby carbodiimide crosslinking to polylysine and targeted to asialoproteinreceptors expressed on cells in the liver. In addition, enhanced nucleicacid delivery and expression can be achieved using a ligand-polylysineconjugate coupled to carbohydrate moieties on viruses that is thencombined with DNA. These preparations are suitable for parenteralinjection and are readily taken up by cells expressing asialoproteinreceptors in the liver after which the DNA is internalized andintegrated into the host genome. In addition, nucleic acid can beadministered intravenously, intramuscularly, or subcutaneously to inducea response in a host.

Thus, targeting nucleic acid to the surface of particular cells isaccomplished by conjugating nucleic acid to molecules that bind to acell surface structure such as a receptor. Examples of cell surfacestructures that can be targeted include, without limitation, thetransferrin receptor, and asialoglycoprotein receptor. The moleculesthat bind cell surface structures and are conjugated to nucleic acid fortargeting can be, without limitation, natural ligands for the surfacestructure, synthetic compositions that exhibit specific binding, andantibodies directed against the surface structure. For example, amonoclonal antibody specific for a cell surface epitope such as the BR96antibody that recognizes Lex carbohydrate epitope abundantly expressedby colon, breast, ovary, and lung carcinomas can be used. Othermonoclonal antibodies can include, without limitation, those thatrecognize growth factor receptors, transferrin receptors, IL-2receptors, epidermal growth factor receptors, the hev oncogene, andTAPA-1 as well as any other antibody having specificity for a surfacestructure that can be internalized.

Liposomes containing nucleic acid are also targeted to specific celltypes such that the nucleic acid is expressed. For example, nucleic acidis loaded into or attached to cationic DOTMA:doleoylphosphatidylethanolamine (DOPE) liposomes that contain exposedmolecules that bind to a cell surface structure such as tumor cells ortumor microvasculature (Example 5). The molecules that bind cell surfacestructures and are attached to liposomes can be, without limitation,natural ligands for the surface structure, synthetic compositions thatexhibit specific binding, and antibodies directed against the surfacestructure. Maximal transfer of nucleic acids encoding SAgs is attainedby synthesizing the liposomes with an appropriate ratio of nucleic acidto lipid. In addition, these nucleic acid- containing liposomes areadministered intravenously, intramuscularly, or subcutaneously to inducea response in a host.

Naked nucleic acid is also administered to a host. For example, nakedpharmaceutical-grade plasmid DNA are injected into a hostintramuscularly such that it is expressed by host cells (U.S. Pat. Nos.5,589,466; 5,580,599; 5,264,618; 5,459,127; and 5,561,064). In addition,cationic lipids are used to deliver biologically active molecules, suchas oligonucleotides to host cells in vivo (U.S. Pat. Nos. 5,264,618,5,459,127, and 5,561,064). Thus, nucleic acid encoding a SAg isadministered to a host in naked or cationic lipid form such that the SAgis expressed. It is noted that any nucleic acid described herein can beadministered in vivo as naked DNA. Further, other methods ofadministering naked DNA to a host can be used such as those related tothe direct injection of naked DNA for use in vaccines (Cohen et al.,Science 259:1691-1692 (1993); Corr et al., J. Exp. Med. 184:1555-1560(1996); Varmus et al., Proc. Natl. Acad. Sci. USA 81:5849-5852 (1984);and Benveniste et al., Proc. Natl. Acad. Sci. USA 83:9551-9555 (1986)).Our previous patent applications which are hereby incorporated byreference include U.S. patent application Ser. No. 07/416,530, filedOct. 3, 1989, U.S. patent application Ser. No. 07/466,577, filed Jan.17, 1990, U.S. patent application Ser. No. 07/891,718, filed Jun. 1,1992, U.S. patent application Ser. No. 08/025,144, filed Mar. 2, 1993,U.S. patent application Ser. No. 08/189,424, filed Jan. 31, 1994, U.S.patent application Ser. No. 08/491,746, filed Jun. 19, 1995, PCTapplications PCT/US91/00342, and PCT/US94/02339. These applications havegiven comprehensive description of the SAg genes, the creation of highenterotoxin producing mutant strains as well as recombinant methods ofproduction of SAgs. In addition, methods of treating cancer bytransfecting tumor cells in vivo and in vitro with SAg nucleotides usingwell defined recombinant technology have been described in theseapplications. Subsequently, Dow et al., (J. Clin. Invest. 99: 2616-2624(1997)) described in vitro and in vivo transfection of eukaryotic cellswith SAg DNA which was capable of inducing inflammatory responses invivo. It is noted that the SAg genes have been cloned and theirsequences delineated before 1988 and methods used to transfect cells invivo or in vitro with nucleic acids encoding polypeptides are also wellknown in the art.

4. Constructs

Tumor cells are transfected with various nucleic acids which aredesigned to increase their immunogenicity and to provide them withcapacity to traffic to metastatic sites where they may initiate a potentinflammatory and immune response. Such constructs of this invention canbe linear or circular nucleic acids obtained from mammals or bacteriathat encode a polypeptide such as a SAg, mutant SAg, erythrogenic toxin,enzymes involved in the biosynthesis of glycosyltransferases, bacterialglycosylceramides, LPS's, lipoproteins, capsular or membranepolysaccharides, microbial toxins and enzymes such as hyaluronidase,collagenase, elastase, coagulase, protease, kinase, lipase. Constructsmay also contain tumor associated antigens, costimulatory molecules suchas B7-1 and B7-2, adhesion molecules, receptor molecules such as SAgreceptors, CD1, CD14, MHC class I molecules and/or MHC class IIreceptors. Such constructs may also contain amplified nucleic acidsassociated with tumors such as oncogenes, transcription factors,angiogenesis factors and receptors, tumor growth factor receptors,chimeric receptors. The latter nucleic acids may be linked toSAg-encoding nucleic acid to produce heightened expression of the SAg.The amplified nucleic acids may include tumor tissue specific promotersand nucleic acids that direct the colonization or metastasis of tumorsto selected sites in vivo. Constructs can also contain elements thatregulate and/or promote the expression of an encoded polypeptide. Forexample, a construct containing nucleic acid that encodes enterotoxin B(SEB) can have a strong promoter element upstream of the SEB encodingsequence. In addition, constructs can contain nucleic acid that anchorsan encoded polypeptide to the cell surface after expression. Forexample, a construct containing nucleic acid that encodes SEB cancontain a membrane-anchoring sequence such as nucleic acid that encodesa hydrophobic stretch of amino acids or a glycosylphosphatidylinositol(GPI)-anchoring motif Thus, the SAg, or other polypeptides as well, canbe anchored in the plasma membrane by coupling to membrane lipids orglycolipids. These anchors can be attached to the C terminus of thepolypeptide in the endoplasmic reticulum. Alternatively, a SAg known tobe associated with the cell surface after expression can be used such asthe mammary tumor viral (MMTV) SAg that is GPI-linked. In oneembodiment, SAgs as well as SAg receptors are engineered to remainanchored to the surface of transfected cells when the cell is to be usedfor immunization. Likewise, when a SAg receptor gene is transfected intoanergized T cells from cancer patients, it is desirable to express thereceptor on the cell surface so that they are readily recognized andactivated by exogenous receptor bound SAg. In contrast, when it isdesirable to use SAg transfected cells to activate T cells in vivo or exvivo or to promote trafficking of transfected tumor cells to metastaticsites in vivo, it is suitable for the SAg to be secreted from thetransfected cells.

In additional embodiments, potent tumor specific effector T or NKT cellclones are produced with overexpressed V□ regions of their TCRs makingthem highly receptive to activation by exogenous SAg. Likewise CD44genes are transfected into T cells or NKT cells making them moresusceptible to expression of this epitope after SAg stimulation.

Constructs also contain a selectable marker or reporter such thattransfected cells can be isolated. For example, a construct containingnucleic acid that encodes a SAg can also contain nucleic acid thatencodes a polypeptide that confers resistance to a selection agent suchas neomycin (also called G418), puromycin, or kanamycin.

Nucleic acid and nucleic acid constructs of the present invention areincorporated into a vector, an autonomously replicating plasmid, or avirus (e.g., a retrovirus, adenovirus, or herpes virus). Typically,these vectors, plasmids, and viruses can replicate and functionindependently of the cell genome or integrate into the genome. Vector,plasmid, and virus design depends on, for example, the intended use aswell as the type of cell transfected. Appropriate design of a vector,plasmid, or virus for a particular use and cell type is within the levelof skill in the art. In addition, a single vector, plasmid, or virus canbe used to express either a single polypeptide or multiple polypeptides.It follows that a vector, plasmid, or virus that is intended to expressmultiple polypeptides will contain one or more operably linkedregulatory elements capable of effecting and/or enhancing the expressionof each encoded polypeptide.

The term “operably linked” means that two nucleic acid sequences are ina functional relationship with one another. For example, a promoter (orenhancer) is operably linked to a coding sequence if it effects (orenhances) the transcription of the coding sequence. A ribosome bindingsite is operably linked to a coding sequence if it is positioned tofacilitate translation. Operably linked nucleic acid sequences are oftencontiguous, but this is not a requirement. For example, enhancers neednot be contiguous with a coding sequence to enhance transcription of thecoding sequence.

A vector, plasmid, or virus that directs the expression of a polypeptidesuch as a SAg can include other nucleic acid sequences such as, forexample, nucleic acid sequences that encode a signal sequence or anamplifiable gene. Signal sequences are well known in the art and can beselected and operatively linked to a polypeptide encoding sequence suchthat the signal sequence directs the secretion of the polypeptide from acell. An amplifiable gene (e.g., the dihydrofolate reductase [DHFR]gene) in an expression vector can allow for selection of host cellscontaining multiple copies of the transfected nucleic acid.

Standard molecular biology techniques are used to construct, propagate,and express the nucleic acid, nucleic acid constructs, vectors,plasmids, and viruses of the invention ((Sambrook, J. et al., supra;Maniatis et al., Molecular Cloning (1988); and U.S. Pat. No. 5,364,934.For example, prokaryotic cells (e.g., E. coli, Bacillus, Pseudomonas,and other bacteria), yeast, fungal cells, insect cells, plant cells,phage, and higher eukaryotic cells such as Chinese hamster ovary cells,COS cells, and other mammalian cells can be used.

Constructs are used in vivo or ex vivo or in combination as in Example5-7, 16-23. They are used to immunize a host by direct in vivoadministration or they are used ex vivo to activate T cells or NKT cellsto become tumor specific effector cells which are employed for adoptiveimmunotherapy of cancer by methods and models (Examples 7, 16, 19-23).

To test the anti-tumor-inducing ability of a particular construct aswell as the transfected cell itself, the following general assay isperformed. B16 melanoma, A20 lymphoma, host tumor cells, or any othertumor cell lines appropriate to the host (i.e., having tumor antigensexpected to be present on the host tumor cells) are transfected with agiven construct. Appropriate numbers of transfected cells (e.g., 10⁵-10⁷cells) are then implanted subcutaneously into animals such as mice,rats, rabbits, or the like and 1-6 months later untransfected tumorcells are implanted. Tumor outgrowth from the untransfected tumor cellsis measured and compared to control animals not given the transfectedtumor cells. If tumor outgrowth is reduced or prevented, then thetransfected cells are effective anti-tumor agents useful as tumorvaccines. Alternatively, 10⁵-10⁷ transfected tumor cells can be given3-10 days after the appearance of established tumors from untransfectedtumor cells. If tumor outgrowth is reduced or arrested, then thetransfected cells are effective anti-tumor agents useful in treatingestablished tumors.

To test the anti-tumor effect of SAg activated T cells, NKT cells or Tcells clones overexpressing V□ or CD44, the following general protocolis used. Lymph node cells from C57/B1 mice bearing MCA 205 or 207sarcomas which were implanted in the adjacent inguinal region three toten days before are extracted and placed in tissue culture. The cellsare incubated with various enterotoxins for two days and then with IL-2for an additional two to three days. The cells are then harvested andinjected into syngeneic mice with established pulmonary metastases (sixto twelve days after tumor injection). Three weeks later the animals areevaluated for pulmonary metastases compared to controls which receive nocells or cells that were stimulated without enterotoxins. The adoptivelytransferred cells may be enriched for NKT cells or T cells alone (toinclude/T cells) which are selectively injected into tumor bearinghosts. Likewise, they are selected for predominant expression of theCD44 phenotype during the SAg activation phase at which time the CD44enriched population is harvested and used for adoptive immunotherapy.The dose of injected T cells, NKT cells or γ/δ T cells and/or CD44enriched cells (which are produced by any of these T cell, NKT cell orγ/δ T cell populations) range from 10⁶ to ⁷ and are be given on aschedule of once weekly for one to four weeks.

5. Superantigens (SAgs)

SAgs are polypeptides that have the ability to stimulate large subsetsof T cells. SAgs include Staphylococcal enterotoxins, Streptococcalpyrogenic exotoxins, Mycoplasma antigens, rabies antigens, mycobacteriaantigens, EB viral antigens, minor lymphocyte stimulating antigen,mammary tumor virus antigen, heat shock proteins, stress peptides, andthe like. Any SAg can be used as described herein, although,Staphylococcal enterotoxins such as SEA, SEB, SEC, and SED andstreptococcal pyrogenic exotoxins such as toxic shock-associated toxin(TSST-1 also called SEF) are preferred.

When using enterotoxins, the region related to emetic activity can beomitted to minimize toxicity. In addition, SAgs can be derivatized tominimize toxicity. The level of toxicity may not be a concern when usingSAg transfected cells to activate lymphocytes ex vivo since thelymphocytes can be rinsed of SAg polypeptide prior to administration toa host.

The nucleic acid sequences that encode SAgs are known and readilyavailable. For example, Staphylococcal enterotoxin A (SEA) (SEQ IDNOS:7-8), SEB (SEQ ID NOS:9-10), SEC (SEQ ID NOS:11-12), SED (SEQ IDNOS:13-14), SEE (SEQ ID NOS:15-16), TSST-1 (SEQ ID NOS:17-18), andStreptococcal pyrogenic exotoxin (SPEA) (SEQ ID NOS:19-20) have beencloned and can be expressed in E. coli (Betley M J and J J Mekalonos, J.Bacteriol. 170:34 (1987); Huang I Y et al., J. Biol. Chem., 262:7006(1987); Betley M et al., Proc. Natl. Acad. Sci. U.S.A, 81:5179 (1984);Gaskill M E and S A Khan, J. Biol. Chem., 263:6276 (1988); Jones C L andS A Khan, J. Bacteriol., 166:29 (1986); Huang I Y and M S Bergdoll, J.Biol. Chem., 245:3518 (1970); Ranelli D M et al., Proc. Nat. Acad. Sci.U.S.A 82:5850 (1985); Bohach G A, Infect Immun., 55:428 (1987); Bohach GA, Mol. Gen. Genet. 209:15 (1987); Couch J L et al., J. Bacteriol.170:2954 (1988); Kreiswierth B N et al., Nature, 305:709 (1983); CooneyJ et al., J. Gen. Microbiol., 134:2179 (1988); Iandolo J J, Annu. Rev.Microbiol., 43:375 (1989); and U.S. Pat. No. 5,705,151)). Additionalnucleic acid sequences encoding SAgs are described elsewhere (Bohach etal., Crit. Rev. in Microbiology 17:251-272 (1990); (Kotzin, B L et al.,Advances Immunology 54: 99-165 (1993)) PCR can be used to isolateSAg-encoding acid. For example, the nucleic acid encoding SEA, SEB, andTSST-1 can be isolated as described elsewhere (Dow et al., J. Clin.Invest. 99:2616-2624 (1997)). Briefly, the following primers can be usedto amplify the SAg-encoding nucleic acid: SEA forward:GGGAATTCCATGGAGAGTCAACCAG, (SEQ ID NO:21) SEA backward:GCAAGCTTAACTTGTTAATAG; (SEQ ID NO:22) SEB forward:GGGAATTCCATGG-AGAAAAGCG, (SEQ ID NO:23) SEB backward:GCGGATCCTCACTTTTTCTTTG; (SEQ ID NO:24) and TSST-1 forward:GGGGTACCCCGAAGGAGGAAAAAAAAATGTCTACAAACGATAATATAAAG, (SEQ ID NO:25)TSST-1 backward: TGCTCTAGAGCATTAATTAATTTCTGCTTCTATAGTTTTTAT. (SEQ IDNO:26)

The full-length TSST-1 nucleic acid sequence is cloned into a eukaryoticexpression vector (pCR3; InVitrogen Corp., San Diego, Calif.), whereasonly the sequence corresponding to the mature SEB and SEA (sequencesminus the putative bacterial signal sequences) is cloned into pCR3.Removal ofthe SEB and SEA signal sequences increases the level ofexpression in transfected cells. The plasmids are grown in Escherichiacoli and plasmid DNA extracted by the modified alkaline lysis method andpurified on a CsCl gradient.

Nucleic acids encoding mutant or variant SAgs are also considerednucleic acid sequences encoding SAgs within the scope of the invention.For example, a mutant SAg-encoding acid sequence is engineered such thatthe resulting SAg is devoid of amino acid residues, e.g., histidine,known to produce toxicity. Likewise, SAg-encoding nucleic acid isengineered to contain or lack sequences that facilitate the selectivebinding of SAgs to certain V□ regions of the TCR present on T cells orto ganglioside, mannose (or other carbohydrate) receptor, certainregions of MHC class II, and/or enterotoxin receptors present on tumorcells, antigen presenting cells (APCs), and/or lymphocytes.

Nucleic acid sequences that encode a SAg are also fused, in frame, withnucleic acid that encodes another polypeptide. This larger nucleic acidis termed herein a SAg fusion gene and the resulting polypeptide productis a SAg fusion product. Nucleic acid sequences that are fused toSAg-encoding nucleic acid include, without limitation, nucleic acidsequences that encode tumor antigens, costimulatory molecules, adhesionmolecules and MHC class II molecules. The superantigen fusion product issecreted by a transfected cell, expressed on the cell surface or it mayremain intracellular in nucleic acid or partly processed form.

SAgs are also isolated and purified from their natural source as well asfrom a heterologous expression system such as E. coli. Likewise,SAg-containing polypeptides (e.g., SAg fusion products) are isolated andpurified from a heterologous expression system. In addition,Staphylococcus strains producing high levels of enterotoxin have beenidentified and are available. For example, exposingenterotoxin-producing Staphylococcus aureus to mutagenic agents such asN-methyl-N-nitro-N-nitrosoguanidine results in a 20 fold increase inenterotoxin production over the amounts produced by the parent wild-typeStaphylococcus aureus strain (Freedman M A and Howard M B J. Bacteriol.,106:289(1971)).

6. Glycosylated SAgs and SAgs Conjugated to Glycosylceramides.Lipopolysaccharides. Glycans and Lipoarabinomannans: Presentation on CD1Receptors for Activation of T or NKT Cells and Differentiation to TumorSpecific Effector Cells.

In a tumor cell or accessory cell, nucleic acid signal sequences areintegrated into nucleic acids encoding the SAg molecules in order toroute them to the Golgi apparatus and endoplasmic reticulum of tumorcells where they are glycosylated via appropriate glycosyltransferases(precedents from the selective transferases used to producemonogalactosylceramide in the Sphingomonas paucimobilis) to produce aproteoglycan with structural similarity to LPS, lipoteichoic acid,GalCer, a Gal, Streptococcus capsular polysaccharide. This construct isthen secreted as an immunogenic “ground substance.” Alternatively, theresulting SAg glycolipid is anchored to the membrane, expressed on thecell surface and routed specifically to CD1 receptors.

SAgs which are glycosylated by the above intracellular processes haveimproved capacity to bind surface structures such as mannose receptors,ganglioside receptors and CD1 receptors. Generally, the nucleic acidsencoding a SAg are modified to include a signal sequence for routing tothe Golgi apparatus and a core sequence which initiates glycosylation.It is important that the V□ TCR binding region is not blocked by theadded carbohydrate modifications. For example, an N-linked glycosylationsite (in the sequence Asn X Ser/Thr where X is any residue except Pro)is engineered into SAg-encoding acid sequences which do not functionallyinterfere with TCR binding and activation. The nucleic acid encodingthese signal sequences and core binding glycosylation sites of SAgs arefused to nucleic acids encoding SAg and the fusion gene used totransfect tumor cells of a host. In addition, glycosylated forms of SAgsare expressed in a heterologous eukaryotic expression system such asyeast cells or baculovirus-infected insect cells. In gram negativebacteria (such as E. coli), nucleic acids encoding SAgs are fused tonucleic acids encoding LPS's, in gram positive bacteria (such asStaphylococcus or Streptococcus), to nucleic acids encoding capsularpolysaccharides and teichoic acids and in mycobacterial species tonucleic acids encoding lipoarabinan.

The gram negative bacterium Sphingomonas paucimobilis produces themonogalactosylceramide. In this bacterium, nucleic acids encoding SAgs(containing serine) are fused to nucleic acids encoding and directingthe synthesis of glycosylceramides and monogalactosylceramide inparticular. The resulting galactosylceramide-SAgs are powerful T cellstimulants. The same procedure is followed in bacteria which naturallyproduce LPS's such as E. coli, Salmonella or Klebsiella or for bacteriawhich naturally produce lipoarabinomannans glycans or polysaccharidescontaining cell walls such as Mycobacterium and Streptococcusrespectively. The SAg-polysaccharide constructs bind to CD1 receptors ofantigen presenting cells. They are then capable of activating NKT cellseither in vivo or ex vivo to become tumor specific effector cells inresponse to IL-12. SAgs are also conjugated genetically or biochemicallyas in Example 5 to LPS's via a natural high affinity binding site forLPS binding protein (LPB). Once bound, the SAg catalyzes the binding ofLPS monomers to CD14 and CD1 receptors in a fashion similar to that ofLPB. In this way, the conjugates are capable of activating T cells foruse in vivo or ex vivo for adoptive immunotherapy while preserving theanti-apoptotic effect of LPS on SAg activated T cells. Examples of theirpreparation and use in vivo and in vitro are given in Examples 4, 7, 15,16, 18-23.

In addition, SAgs similarly conjugated to lipoarabinomannans and glycansare integrated into lymphomonocytic cell membranes viaglycosylphosphatidylinositol anchors. These SAg-lipoarabinomannancomplexes are expressed or secreted by antigen presenting cells or tumorcells. They are also bound to CD1, mannose or class II receptors inwhich form they are used to activate T or NKT cells. These constructsare administered in vivo or they are used ex vivo to produce tumorspecific effector cell populations (T cell or NKT cells) which areemployed for adoptive immunotherapy of cancer (Examples 5, 15-16,18-23).

Mannose receptor expression is upregulated by cytokines. For example,accessory cells including DCs, and tumor cells express mannose receptorson their surfaces after GM-CSF treatment. SAgs are bound to mannosereceptors by transfecting cells with nucleic acids encoding SAg whichalso consist of nucleic acids encoding signal sequences andglycosylation sites which, in the presence of appropriateglycosyltransferases, produce mannosylated SAgs. These preferentiallybind to mannose receptors. In addition, glycosylated SAgs bind toamphipathic cell surface gangliosides and glycolipids via hydrophobicinteractions. These glycosylated SAgs presented in a form bound tomannose receptors are capable of activating T cells and NKT cellpopulations. They are used either in vivo by direct administration or exvivo to produce a tumor specific effector cell population (T cell or NKTcells) for use in adoptive immunotherapy of cancer (Examples 4, 5, 15,16, 18-23).

7. SAgs Conjugated to Glycosylceramides, Gangliosides and Verotoxins(VT)

Amphipathic ganglio sides bound to tumor cell surfaces such as GD1, GD2,GD3, GM1, GM2, GM3, GQ1 and GT1 are capable of binding exogenous SAgs.The binding of a SAg to the surface of a tumor cell creates an immunogenon the tumor cell surface. Tumor cells transfected with nucleic acidsencoding glycosyltransferases overexpress gangliosides, producing agreater surface density of ganglioside moieties available to bindexogenous SAgs. Enterotoxins bind to cell surface amphipathicgangliosides and/or glycophorins via their hydrophobic residues whilepreserving their T cell binding properties. SAgs are also glycosylatedintracellularly by addition of a glycosylation site or by chemicalconjugation of a carbohydrate moiety using methods well described in theart. In glycosylated or native form, the SAgs bind to surfaceganglioside while retaining their T cell activating properties.Overexpression of the hydrophobic regions of the molecule promotesbinding to the surface gangliosides (Example 5). Examples from nature ofexogenous proteins that bind to cell surface gangliosides includefalciparum malarial merozoite which combines with gangliosidesassociated with the Duffy blood group and induce long standing anddurable protection and tetanus toxin which binds to surface gangliosideswith highest affinity for the disialyl groups linked to inner galactosylresidues.

Enterotoxin B contains a T cell activating sequence which is chemicallycross-linked or polymerized using bifunctional agents such ascarbodiimide, glutaraldehyde or formaldehyde by established methods wellknown in the art. These polymers are then bound to gangliosidesexpressed on tumor cells such as GD1, GD2, GQ1, GD3 or GM1, GM2, GM3,GT1. In monomeric or polymerized form, SAgs also bind tomonogalactosylceramides which are free or bound to CD1 receptors ontumor cells or antigen presenting cells via hydrophobic interactions.The monogalactosylceramide binds to hydrophobic sequences on the SAgwhich are expressed at multiple sites on the molecule. In oneembodiment, the lauroyl group [CH₃(CH)₁₀CO] or the group [CH₃(CH)₁₃CO]is covalently added to each of the peptide's amino terminus to serve asa of the CD1 receptor. The key SAg peptide sequence such as of SEB(amino acids 225-234) which confers T cell activating properties istandemly repeated to various lengths prior to lipid conjugation.

Hydrophobic SAg peptides(such as Trp, Tyr, Phe, Leu, and Ile) arescreened for binding to glycosylceramides immobilized on CD1 receptorsor via adsorption chromatography with immobilized glycosylceramide. TheSAg sequences with the greatest affinity for the CD1 receptor areselected for conjugation to the glycosylceramides and LPS's.Alternatively, the SAg sequence is screened for affinity for the CD1 orMHC class II receptor using a peptide phage display library as describedin Examples 4. Likewise, pre-formed SAg-glycosylceramide or LPScomplexes are also screened for affinity for the CD1 or MHC class IIreceptor (Example 4). These lipopeptide complexes are then screened forT cell proliferative activity and IL-12 production. The monomeric orpolymerized SAg in native or glycosylated form binds to themonoglycosylceramides or gangliosides expressed on CD1 receptors on thetumor cell surface.

Therapeutic Construct: SAg-Glycosylceramide Conjugates

SAgs have an affinity for glycosphingolipids especially those withterminal or subterminal Gal(α1-4)Gal residues. Such residues areexpressed on tumor cells as Gal(α1-4)Gal(□1-4)GlcCeramide(globotriaosylceramide or Gb3) and Gal(α1-4)GalCeramide(galabiosylceramide or Gb2). Gb3 and Gb2 also known as CD77, Burkitt'slymphoma antigen, and the human blood group p^(k) antigen are thenatural receptors for Shiga toxins and VT's . Shiga toxin, a 69-kDacomplex of proteins comprised of five □-subunits (7 kDa each) and onea-subunit (30 kDa) has high affinity for the terminal digalactose of Gb3or Gb2. Methods for their preparation and isolation are described inExample 41. Once bound to the tumor cell, these toxins are internalizedand induce apoptosis.

The synthetic pathway for neutral glycosphingolipids in eukaryotic cellsis known. Glucosylceramide (GlcCer) is the precursor of lactosylceramide(LacCer), which leads, in order, to Gb3 and globotetraosylceramide(Gb4). Different Golgi enzymes are responsible for addition ofmonosaccharides from nucleotide-sugar donors in each step of thepathway. Globotriaosylceramide synthase (UDP-galactose:lactosylceramideα1-4-galactosyltransferase) has been purified. In the cytoplasm, theα-subunit of the Shiga toxin or VT is processed by a trypsin-likecleavage. The “activated” 27-kDa α-subunit inactivates 60S ribosomes bydepurination of a single nucleotide in 28S rRNA, rendering ribosomesincapable of carrying out peptide elongation.

The present invention provides therapeutically active soluble complexescomprising SAg and glycosphingolipids which have terminal or subterminalGal(α1-4)Gal residues and Shiga toxin receptors Gb3 and Gb2,(collectively referred to as “GTSG1-4”). These complexes include but arenot limited to SAg-GPI-GTSG1-4 complexes, and synthetic and functionalderivatives thereof. Such structures appear naturally on surfaces ofcertain tumor cells such as astrocytoma, Burkitt's lymphoma and ovariancarcinoma. Methods of preparing and isolating glycosylceramides and VTsare given in Example 41.

SAgs also have a demonstrable affinity for galactosylceramidescontaining Gal(α1-4)Gal residues. Methods of assessing SAg binding toGTSG1-4 are provided given in Example 43. These conjugates are also shedfrom SAg-transfected tumor cells as binary complexes of SAg-GTSG1-4 orternary complexes of SAg-GPI-GTSG1-4, in free form, as vesicles or asexosomes(see Sections 38 and Example 38). Methods of isolating andcharacterizing these shed complexes appear in Section 38 and Example 42.The complexes may also be prepared by chemical or genetic methods(Example 5). SAg-GTSG1-4 or SAg-GPI-GTSG1-4 complexes or exosomes areuseful as a preventative vaccine or against established tumor. They arealso useful in vivo by direct administration or ex vivo where they areloaded onto antigen presenting cells comprising CD1 or MHC receptors toactivate NKT and T cells to produce tumor specific effector T or NKTcells for adoptive therapy of cancer (Examples 5, 7, 14, 15, 16, 18-23,38).

Therapeutic Construct: Tumor Cells Expressing SAgs andGalactosylsylceramides

Additional immunogenic complexes comprising SAgs bound to tumor cells,DCs DC/tc constructs expressing surface Gb2 and Gb3 or otherglycosphingolipids containing terminal Gal(α1-4)Gal are prepared bytransfecting these cells with nucleic acids encoding a SAg. Thetransfected cell expresses the SAg in the context of theglycosphingolipid comprising the terminal or subterminal Gal(α1-4)Galmoiety. Alternatively, free or GPI linked glycolipids containing SAgpeptides or polypeptides bind to tumor cells or accessory cells intissue culture (Section 38). The expression of Gb3 and Gb2 on tumorcells is optionally upregulated by various cytokines, including IFNα andTNF α, before contacting the SAg

Tumor cells, accessory cells or fused tumor/accessory cells transfectedwith SAg which are not naturally endowed with the GalCer (optionallycoupled to SAg) acquire these molecules in free or GPI-linked form fromsurrounding media or by transfer from liposomes or vesicles (exosomes)which express them (Section 38 and Example 5). The resulting cells,coexpress SAgs and glycosylceramides or other glycosylceramides capableof stimulating an effective T or NKT cell immune response. Multidrugresistant (MDR) tumor cells or cell lines which naturally accumulate andexpress intracellular glycosylceramides are useful in this invention.MDR agonists such as SDA PSC 833, a cyclosporin analogue, and fumonisinB1, a ceramide synthase inhibitor, are employed to induce ceramideaccumulation in MDR cells (Example 45). Tumor cells or accessory cellswhich overexpress key glycosylceramides due to transfection with α□-2,α1-4, α1-6 glycosyltransferases (Example 38) or a natural or induceddeficiency of α-galactosidase are also useful. In addition, tumor cellswith high concentrations of GalCer expressed on their surface or that ofaccessory cells are generated by incubation with ceramides containing a2-hydroxy fatty acid C₆OH. Tumor cells selectively convert them toGalCer, galabiosylceramide and sulfatide in the trans-Golgi networkwhere they are sorted and transported selectively to the cell surface.Methods for this selective biosynthesis of GalCer with hydroxy fattyacids are in Example 46.

These fused SAg-tumor cell/accessory cell constructs are used toactivate a T or NKT cell population. They are used in vivo by directadministration or ex vivo to produce a population of tumor specificeffector cells (T cells or NKT cells ) for adoptive therapy of cancer(Examples 5, 7, 14, 15, 16, 18-23, 38).

SAg-VT Conjugates to Induce Tumor Cell Apoptosis

The present invention contemplates the induction of apoptosis in tumorcells expressing Gb2 and Gb3 (or other glycosphingolipids containingterminal Gal(α1-4)Gal) by using free SAgs, conjugates and fused DNA thatcomprises SAg, SAg peptide or SAg-encoding DNA fused to intact VT or toVT A or B chains. Preparation of these conjugates and fusion proteinsfrom their corresponding DNA, polypeptides or functional derivatives isprovided in Examples 1 and 5. These conjugates induce apoptosis bybinding to tumor cell glycosphingolipid receptors having terminalGal(α1-4)Gal. Methods of assessing tumor cell apoptosis are in Example44. CD19 or IFNα peptide sequences and generic carbohydrate recognitiondomains which bind Gal(α1-4)Gal structures are also useful. CD19, aB-cell restricted differentiation antigen, naturally binds to Gb3 andGb2 on the cell surface which incudes apoptosis. CD19 has VT-likesequences in the N-terminal extracellular domain (NBRF protein databank) that have 41%, 34% and 37% sequence identity to VT1, VT2, and VT2eB subunits, respectively. When compared to a consensus VT B sequence,the CD19 sequences show 49% identity. Binding of these peptide sequencesto membrane Gal(α1-4)Gal-containing glycolipids facilitates receptormediated induction of apoptosis.

The IFNα receptor has a 63-kDa extracellular peptide with regions ofamino acid identity to domains in the VT B subunit implicated as Gb2/Gb3binding sites. The preferred targets of the above conjugates on tumorcells are the naturally expressed Shiga toxin receptors Gb3 and Gb2 witha terminal Gal(α1-4)Gal. Astrocytomas and Burkitt's lymphomas are thepreferred tumors as they naturally express glycosphingolipid receptors.However, any tumor expressing the appropriate receptor is appropriate.Tumor cells which express either engineered or natural functionalderivatives, or mutants of these glycosphingolipid receptors, are alsouseful. Receptor expression on the target cells is optionallyupregulated by cytokines such as IFNγ and TNFα. Tumor cell sensitivityto the cytotoxic effects of a VT is enhanced by administration ofinterleukin-1□ before the addition of the conjugates. Tumor cells whichdo not naturally display Gb3 or Gb2 acquire these structures by transferfrom free, soluble structures or liposomes which express the missingglycosphingo lipid receptor (Section 38, Example 5). The reconstitutedtumor cells bearing the appropriate glycolipid receptors are thustargeted for apoptosis by the above constructs and conjugates.

SAg Nucleic Acid-Verotoxin Conjugate

A preferred construct is the SAg-VT conjugate wherein the SAg ispreferably in nucleic acid form (prepared according to Example 3). TheVT portion of the complex binds to the tumor cell and initiatesapoptosis. The VT also acts as a “vector” for transfer of the SAgnucleic acid into the cell. SAg-VT conjugates bind to the terminalGal(α1-4)Gal receptors on tumor cell surfaces and are internalized viaendocytosis. The SAg nucleic acid is internalized together with the VT.The VT A chain is an RNA N-glycosidase acting on the 60S ribosomalsubunit. It induces apoptosis in the tumor cell by removing an adeninebase on amino acyl-transfer RNA so that peptide chain elongation isblocked. The resulting apoptotic tumor cells contain the internalizedSAg nucleic acid and are then ingested by dendritic cells. The DCs arecross primed to induce an effective anti-tumor response by presentingthe tumor associated antigens in the class I pathway to T cells whilethe SAg nucleic acid expresses SAg polypeptide. These activated DCs orDC/tc hybrids can be prepared by the methods of Examples 28-29. They areused to activate a T or NKT cell population in vivo as a preventativevaccine or by direct administration against established tumor. They arealso used ex vivo to produce a population of tumor specific effectorcells (T cells or NKT cells ) for adoptive therapy of cancer (Examples5, 7, 14, 15, 16, 18-23, 28-29).

Glycosylation or lipid binding of the enterotoxin does not interferewith T cell binding and activating properties. The SAg is glycosylatedby chemical or recombinant techniques described in the Examples 4. TheSAg glycoprotein is the further conjugated to gangliosides in theganglioside synthetic pathway via the presence of key signal peptides onthe glyco-SAg (Example 4). The SAg is also rerouted to the LAMP pathway,glycosylated in the Golgi apparatus and the endoplasmic reticulum andthen translocated to the membrane class II receptor as a glycosylatedganglioside. Gangliosides are glycosylated to form glycosylceramides byrecombinant techniques as described in the Example 4. They are alsoglycosylated by glycosyltransferases to form homologues which bind tohydrophobic regions of the SAg peptide. The final products namelySAg-glycosylceramides or SAg-LPS's then bind to CD1 receptors and areused to activate T cells or NKT cells. These construct are administereddirectly vivo or they are useful ex vivo to produce a population oftumor specific effector T cells or NKT cells for adoptive immunotherapyof cancer by protocols given in Examples 7, 15, 16, 18-23).

The present invention contemplates the fusion or coexpression within thesame cell of SAg polypeptides with anomeric mono anddigalactosylceramides which are expressed within a tumor cell or on thetumor cell surface. These construct could also be effectively expressedon the surface of accessory cells defmed in Oxford Dictionary ofBiochemistry and Molecular Biology 1997 edition as any one of varioustypes of cell which assist in the immune response cell and includes butis not limited to DCs, fibroblasts, synoviocytes, astrocytes antigenpresenting cells, neutrophils, macrophages, basophils, eosinophils, mastcells, keratinocytes and platelets, as well as fusion cells comprisingaccessory cells and tumor cells.

The anomeric mono and digalactosylceramides have been shown to activateNKT cells and to produce an anti-tumor response in the context of IL-12.The galactosyl ceramides have several structural requirements in orderto produce anti-tumor effects. 12. Mono and digalactosylceramidesrequire an anomeric galactose or glucose as the terminal sugar or innersugar as for example anomeric 1,6-digalactosylceramide, -anomeric1,2-digalactosylceramide, anomeric 1,4-digalactosylceramide, adiglycosylceramide wherein the inner sugar is an anomeric galactose oran anomeric glucose and anomeric galactosyl or anomeric glucosylceramide. In addition, the 3- and 4-hydoxyl groups on thephytosphingosine portion of the ceramides are preferably unsubstituted,the sphingosine base length is preferably from about 10 to about 13carbon units and the fatty acyl chain length is preferably in the rangeof about 12 to about 24 for optimal anti-tumor effectiveness of themolecule.

The expression of anomeric mono- and digalactosylceramides in a cell isachieved by several methods. The first involves the transfection andamplification of nucleic acid encoding the enzymes which synthesize theanomeric 1,4-, the anomeric 1,6- or the anomeric 1,2.- mono- anddigalactosylceramides such that these glycolipids are overproduced. Thegenes for these transferase enzymes have been cloned. Transfection ofnucleic acid encoding these terminal transferases into the above cellsis carried out in vivo by the methods described in Example 1.

A second method for creating cells that overexpress the foregoingglycolipids uses monensin or brefeldin which block additionalglycosylation and sialylation of the -galactosylceramides, so that themono- and digalactosylceramides accumulate in the cell.

A third approach employs cells from patients with Fabry's disease. Thesecells are genetically deficient in the -galactosidase so they naturallyaccumulate -galactosylceramides.

In a forth technique, an -galactosidase deficiency is induced in thetarget cell so that -galactosylceramides accumulate.

In a fifth approach, the -galactosyltransferase is transfected Fabry'sdisease cells, thereby adding to the usual accumulation due to thecatabolic enzyme deficiency. Such cells should have massiveaccumulations of -galactosylceramides.

In a sixth approach, the desired mono- or diglycosylceramide expressedon liposome surfaces are transferred to tumor cells lacking thesestructures by co-culture and employment of fusion techniques given inexample 5.

Nucleic acids encoding SAgs are transfected into the above cells whichare overexpressing, overproducing or otherwise accumulating mono anddigalactosylceramides. The Golgi apparatus (or Golgi complex) is a majorsite of synthesis of the foregoing glycolipids. In the present context,the SAg combines with it the mono and digalactosylceramides. From theGolgi the SAg-galactosylceramide conjugates or complexes, with theappropriate sorting signals, are dispatched in transport vesicles toother destinations. For a SAg peptide to combine effectively with an-galactosylceramide, the peptide must first have the appropriate sortingsignal which directs it to the Golgi and from there, after complexingwith the glycolipid, to the cell surface. The trafficking pathway of SAgpolypeptide from the ER to the Golgi does not require special signals.SAg polypeptides that enter the ER (and fold and assembles properly)will automatically be transported through the Golgi apparatus to thecell surface unless they carry signals that either detain them in anearlier compartment en route or divert them (via the Golgi apparatus) tolysosomes or secretory vesicles. The SAg-glucosylceramide conjugates arerouted from the Golgi to the cell surface after acquiring a structurelike a cytoplasmic tail such as phosphoinositol which assures that thesemolecules will be bound in the cell membrane. The conjugates may also berouted to CD1 or MHC class I receptors, or via, the class II pathway, toMHC class II receptors by associating with invariant chain or LAMP-1signals as described in Section 8.

The mono- and digalactosylceramides are capable of stimulating NKT cells(via an invariant chain) in the presence of IL-12 to produce ananti-tumor response. SAgs are capable of stimulating a T cell-dependentanti-tumor response. The present invention utilizes tumor cells,accessory cells or hybrid cells such as DC/tc, engineered to expressSAg--galactosylceramide for anti-tumor therapy. These cells may beadministered as a preventative or therapeutic vaccine (Example 29).Alternatively, they may be useful ex vivo to activate an NKT or T cellpopulation for use in adoptive immunotherapy of cancer (Example 29).

8. SAg Targeting to Lysosomes

LAMP-1 is a transmembrane protein localized predominantly to lysosomesand late endosomes. The cytoplasmic domain of LAMP-1 contains the aminoacid sequence (SEQ ID NO:29) Tyr-Gln-Thr-Ile whose structure conforms tothe Tyr-Xaa-Xaa hydrophobic amino acid motif that mediates cell membraneinternalization and possibly lysosomal targeting of several surfacereceptors. The intracellular targeting of LAMP-1 is controlled by theTyr-Gln-Thr-Ile motif located at the C terminus of its cytoplasmic tail.

In the present invention, nucleic acid encoding a SAg is fused withnucleic acids encoding the transmembrane and cytoplasmic tail of LAMP-1.Nucleic acids encoding the signal peptide (N terminal) of LAMP-1 areintegrated into this chimeric construct. These chimeric SAg/LAMP-1polypeptides are targeted to endosomal and lysosomal compartments,thereby rerouting transfected SAg polypeptides into the MHC class IIprocessing pathway. Thus, cells such as tumor cells transfected withnucleic acid encoding this modified SAg preferentially target the SAg tolysosomal compartments and are presented to T cells in the context ofMHC class II. MHC class II negative tumor cells are also transfectedwith nucleic acid encoding MHC class II molecules. The association ofSAgs with MHC class II molecules, their natural ligands on APCs, produceoptimal T cell activation to the tumor. Antigen presenting cellstransfected with these constructs are capable of inducing potentactivation of T cells. Tumor cells, in particular, transfected with thisconstruct are administered directly in vivo or used ex vivo to sensitizea T cell population which is useful in adoptive immunotherapy of cancerby protocols described in Example 16, 18-23).

10. SAg Receptors

It is clear that certain tissues express receptors for enterotoxins thatare not MHC class II and that binding is reserved for selectedenterotoxins and not others. Non MHC cell II binding has been reportedfor colon carcinoma, mast cells epithelial cells and B cells. In a tumorbearing patient, it is desirable for administered SAgs to target tumorcells in vivo. which naturally express enterotoxin binding sites orreceptors. Natural ligands for these receptors are native enterotoxins.However, because of the existence of naturally occurring enterotoxinspecific antibodies in the circulation, native enterotoxins areincapable of binding target tumor cell or T cells. The isolated receptoris used to screen and identify SAg proteins and/or nucleic acids whichbind to the native or chimeric receptor. SAg constructs are producedwhich target the tumor via its SAg receptor while also retaining T cellactivating properties. In addition, T cells or NKT cells from tumorbearing patients are anergized in the course of tumor growth and areincapable of being used as a source of T cells for ex vivo stimulationand adoptive immunotherapy. After transfecting these cells with nucleicacids encoding enterotoxin receptors, they are capable of responding toexogenous enterotoxins and are once again a source of T cells useful inadoptive immunotherapy of cancer by protocols given in Examples 8, 9,12, 16, 18-23.

Methods for receptor isolation purification and retrieval of cDNA aregiven in Example 12. The nucleic acids encoding SAg receptors aretransfected into cells by methods given in Example 1 Tumor cells have anatural binding site for exogenously administered SAg polypeptides. Inaddition, nucleic acid encoding the SAg receptors are transfected into Tcells, NKT cells, or γ/δ T cells of cancer patients which have beenanergized in the course of tumor growth. The expression of the SAgreceptor permits these cells to proliferate and produce TH1 cytokines inresponse to exogenous native SAg, Hence, these autologous T cellpopulations are useful in adoptive immunotherapy. Likewise, accessorycells are transfected with SAg receptor genes and used ex vivo topresent SAg to T cells. Further, the nucleic acid encoding the SAgreceptor is transfected into T cells and fused, in frame, to the nucleicacid encoding the TCR-associated ζ chain or the IL-2 γ to produce achimeric receptor capable of generating a signal for cell proliferationand the release of TH1 cytokines after binding its natural ligandexogenous SAg.

In one embodiment, the enterotoxin receptor is immobilized as in Example12 and used to screen oligonucleotide libraries for binding (Gold L, J.Biol. Chem. 270:13581-13584 (1995)). Avidly binding oligonucleotides areused to mimic the native enterotoxin by targeting the receptor in vivo.They are coupled to the TCR binding site of an enterotoxin peptide. Inthis way, the hybrid molecule is administered to the patient in a formprotected from circulating enterotoxin-specific antibodies.Additionally, a nucleic acid molecule is prepared which mimics theenterotoxin in its ability to bind to the enterotoxin receptor on tumorcells and to the TCR on T cells. This nucleic acid mimicking the nativeenterotoxin is administered to the tumor bearing patients and is capableof targeting the enterotoxin receptor sites on tumor cell and the TCRwithout being eliminated by circulating enterotoxin specific antibodiesas in Example 13, 18, 20-23.

11. Tumor Cells that Express SAgs and the αGal Epitope

Tumor cells are for the large part weakly antigenic and poorlyrecognized by the immune system. various attempts to increase theimmunogenicity of tumor cells by transfection of various cytokines orhistocompatibility antigens have for the most part been unsuccessful.Hyperacute rejection of xenografted organs is a very rapid and dramaticimmune event often occurring within minutes of vascularization of thexenografted organs. Very recently, a major antigenic system onxenografts which is the target of this reaction has been identified asαGal□1-3Gal□1-4GlcNAc or αGal. This epitope is expressed in the tissuesof pigs, guinea pigs, rodents, dogs, and cows but has not been detectedin human tissue. The present invention improves the antigenicity oftumor cells and their recognition by the immune system by providing theGal epitope on the cell surface either alone or together with SAgexpression.

The αGal epitope is expressed by endothelial cells in xenografts such aspig organs is a major antigenic target causing hyperacute organrejection in human transplant patients. This hyperacute rejectionappears to involve a complement dependent mechanism that occurs within afew minutes. An α1-3-galactosyltransferase, is an enzyme capable ofproducing α1-3-galactose-□1-4-N-acetylglucosamine moiety by adding aterminal galactose residue to a subterminal galactose residue via anα1-3 linkage. In addition, the α1-3-galactosyltransferase is notexpressed by human and certain primate cells. Humans containxenoreactive natural antibodies that recognize Gal. For example,anti-Gal antibodies bind to pig endothelial cells that express the Galepitope. These anti-Gal antibodies are naturally occurring IgMantibodies recently found to be present in large amounts in human serum.Surface expression of the αGal epitope on tumor cells is achieved bytransfecting a cell with a cDNA clone encoding theα1-3-galactosyltransferase. While tumor cells are the preferred cellsfor transfection, other cells such as accessory cells or immunocytes arealso contemplated as being within the scope of this invention.

Nucleic acids encoding α1-3-galactosyltransferase polypeptides are known(Sandrin, M S et al., Proc. Natl. Acd. Sci. U.S.A 90: 11391-11395(1993)). A cDNA clone encoding murine 1-3-galactosyltransferase isprepared using the known sequence of this protein and the polymerasechain reaction (PCR) technique (Dabrowski, P L et al., Transplant. Proc.26: 1335-1337 (1994). Briefly, two oligonucleotide primers aresynthesized: (SEQ ID NO:30) 5′-GAATTCAAGCTTATGATCACTATGCTTCAAG-3′, whichis a sense primer that encodes the first 6 amino acids of the mature1-3-galactosyltransferase and contains an HindIII restriction site; and(SEQ ID NO:31) 5′-GAATTCCTGCAGTCAGACATTATTCTAAC-3′, which is ananti-sense primer that encodes the last 5 amino acids of the premature1-3-galactosyltransferase and contains an in-frame termination codon andPstI restriction site. These primers amplify a 1185 bp fragment from aC57BL/6 spleen cell cDNA library that is subsequently purified, digestedwith HindIII and PstI (Pharmacia LKB) restriction endonucleases, anddirectionally cloned into HindIII/Pst I-digested expression vector suchas CDM8 vector. After verifying the correct sequence, the1-3-galactosyltransferase-containing expression vector is transfectedinto heterologous cells such as COS cells to confirm activity. Activitycan be confirmed by testing transfected cells for Gal expression usingthe IB4 lectin (Sigma) of Griffonia simplicifolia that binds to Galresidues.

In the preferred mode, cells transfected with nucleic acids encoding aSAg are co-transfected with nucleic acids that encode an-galactosyltransferase. Alternatively, nucleic acids encoding thetransferase are transfected into a separate cell population which iscoadministered with the SAg transfected cell population.

The SAg-encoding nucleic acid can be transfected into cells whichalready express Gal epitope. In addition, any cell can be transfectedwith the -galactosyltransferase-encoding nucleic acid. For example,Gal-negative human tumor cells or tumor cell lines such as melanoma oradenocarcinoma are transfected with nucleic acid encoding the-galactosyltransferase. Tumor cells transfected with-galactosyltransferase-encoding nucleic acid express the Gal on theirsurface and are rapidly rejected when administered to a host withpreexisting Gal specific antibodies. Methods of transfection are givenin Example 1. Human tumor cells expressing the Gal epitope aftertransfection, become strongly reactive with human serum containingpreexisting antibodies to the Gal epitope.

Thus, an Gal-expressing tumor cell is rejected after implantation. Theability of Gal-transfected tumor cells to induce rejection isdemonstrated by implantation into severely compromised immune deficient(SCID) mice that have been reconstituted with human T and B cells andtransfused with normal human plasma containing the naturally occurringhuman antibodies specific for the Gal epitope. In this case, tumor cellstransfected with -galactosyltransferase-encoding nucleic acid isrejected while untransfected cells are not. Similarly, tumor cellstransfected with -galactosyltransferase-encoding nucleic acid isrejected when implanted into species such as humans which synthesizeantibodies to the Gal epitope compared to untransfected control tumorcells that are unaffected by the treatment.

For example, pretreatment with 10⁵-10⁷-galactosyltransferase transfectedtumor cells subcutaneously followed by implantation of untransfectedtumor cells prevents the outgrowth of untransfected malignant tumorcells. Hence, the -galactosyltransferase transfected tumor cellsfunction as a vaccine. Further, -galactosyltransferase transfected cellsimplanted into animals after untransfected tumors are established inducerejection of an established untransfected tumor.

To test for the presence of Gal on a cell surface, 1-3galactosyltransferase knockout mice that do not express the Gal antigenare used. The 1-3 galactosyltransferase knockout mice are describedelsewhere (Tearle et al., Transplantation 61:13-19 (1996) and Shinkel etal., Transplantation 64:197-204 (1997)). A syngeneic tumor cell that isGal negative such as B16 melanoma variants is transfected with nucleicacids that encode a given carbohydrate modifying enzyme. Thesetransfected cells are then implanted into the knockout mouse thatreceived plasma containing Gal specific antibodies. Tumors do not growin animals containing Gal specific antibodies if the Gal epitope isexpressed. Thus, hosts implanted with Gal positive tumor cells exhibitless growth than those exhibited in hosts implanted with tumor cellsthat are Gal negative.

Gal negative transgenic animals are prepared which are useful fortesting Gal expressing tumors. To produce these animals, nucleic acidsencoding Gal fucosyltransferase are transfected into Gal positive mice.The fucosyltransferase dominates the usage of substrateN-acetyllactosamine and precludes -galactosyltransferase from utilizingthis substrate. The transgenic mice do not express -Gal on the cellsurface. In this way, transgenic mice with the H antigen rather than theGal antigen develop. Transgenic guinea pigs producing minimal Gal arealso created in this way. These animals are used as models for testingtheir capacity to reject syngeneic Gal positive tumors. These systemsalso permit the testing of Gal specific antibodies for anti-tumoreffects after they are passively infused into animals bearing Galpositive tumors.

Neuroblastoma and some melanoma cells overexpress severaldisialogangliosides, for example, GD2 and GD3. In the present invention,nucleic acid-encoding specific sialidases or glucosidases orneuraminidases that cleave terminal sialic acid or carbohydrate residuesare transfected into cells that then express or overexpress aganglioside with an exposed Gal epitope.

Fucosylated glycolipids such as B group antigens, Lewis blood groupantigens, and L-selectin ligands are converted to the a_(i)Gal epitopeusing the appropriate sialidases and glycosyltransferase enzymes. Forexample, a desialylating enzyme is introduced into B group antigenexpressing cells such that the -1,3-linked galactose is exposed and nowrecognized by Gal antibodies. Mild acid treatment to remove thebranching fucose residues on the fucosylated B antigen is used to exposethe

,3 galactose residues. Alternatively, cells expressing the B antigen orselectin antigen are transfected with a,-galactosyltransferase-encodingnucleic acid that competes successfully with fucosyltransferases forN-acetyl-lactosamine substrate and preferentially expresses the aêGalepitope

Nucleic acid encoding other polypeptides are also used to produce thesurface expression of the Gal epitope such as nucleic acid encodingglycosidases that specifically cleave carbohydrate residues to exposethe Gal epitope. Tumor cells transfected with nucleic acids encodingN-acetyl-glucosaminyl transferase show an increased tendency tometastasize and colonize new organs. These same tumor cells arecotransfected with nucleic acids encoding SAgs, Staphylococcalhyaluronidase and erythrogenic toxins as well as Streptococcal capsularpolysaccharide which enables them to secrete enzymes and toxins locallyinducing a potent inflammatory and immune response at metastatic sites.Co-transfection of tumor cells with nucleic acid encoding SAg andnucleic acid encoding a galactosyltransferase, sialidase, and/orglycosyltransferase results in expression of SAg, GalCer, Gal, or otherglycolipids on the cell surface. These tumor cells are used to stimulateT or NKT cells ex vivo to produce a population of tumor specificeffector cells which are deployed for adoptive immunotherapy of cancer.

Mutation of the glycosyltransferase nucleic acid in tumor cells producesa specific LPS containing the Gal/Cer or the Gal which coordinated withgenes for protein glycosylation produce the desired integrated SAg LPS.

13. Tumor Cells Expressing SAgs, Glycosylceramides and LPS's and theirReceptors

It appears that anti-tumor responses are produced by a subpopulation ofT cells known as NKT cells. These cells recognize glycosylceramides withcertain specifications which are presented in the context of CD1receptors on antigen presenting cells. They produce IL-12 mediatedanti-tumor responses. Peptides of certain length with hydrophobicsequences have been shown to react with various hydrophobic regions ofthe CD1 receptor and produce an immune response. However, these peptideshave not been implicated in an anti-tumor response. In the presentinvention, lipoproteins are contemplated which consist of SAg or theirmajor bioreactive domains fused to glycosylceramides in the context ofthe CD1 receptor.

To make this construct, CD1 positive cells are transfected with nucleicacids encoding glycosyltransferases that result in GalCer or GlcCerexpression on the cell surface and preferably in the context of the CD1receptor. The appropriate glycosyltransferase nucleic acid is obtainedfrom Sphingomonas paucimobilis or Agelas mauritianus which are known toexpress the GalCer on their cell surface. The GalCer and GlcCer moietiesare recognized by NKT cell Va invariant chains in the context of CD1receptors on antigen presenting cells. CD1 positive cells arecotransfected with nucleic acids encoding SAgs The resulting CD1positive cells coexpress both GalCer and SAg on the cell surface or inthe context of CD1. The GalCer and SAg presented simultaneously as acomplex and/or separate from each other on the cell surface, in thecontext of CD1 produces potent activation of NKT cells due torecognition of SAg by NKT cell V□ chain and GalCer by the Vα invariantchain. Such GalCer-SAg complexes are loaded onto the CD1 receptor andpresented to NKT cells in this fashion. A SAg peptide capable of bindingto the TCR and activating the T cell is useful for coupling to theGal-Cer before or after it is positioned on the CD1 receptor. (SeeExamples 1-4, 5). CD1 positive antigen presenting cells or tumor cellsbearing the SAg glycosylceramide are used to stimulate a population ofNKT cells ex vivo which is then useful in adoptive immunotherapy ofcancer by protocols given in Examples 7, 15, 16, 18-23). They are alsouseful when administered directly in vivo to tumor bearing patients toproduce an anti-tumor response. (See Examples 18-23).

In the present invention, nucleic acids encoding the CD1 receptor aretransfected into tumor cells in vivo or ex vivo. Martin L H. et al.Proc. Natl. Acad. Sci. U.S.A 83: 9154-9158 (1986). Nucleic acidsencoding the CD1 receptor are also cotransfected into tumor cells withnucleic acids encoding the SAg receptor. A tumor cell expressing achimeric receptor comprising sequences of CD1 and SAg receptors is alsoproduced by transfection of fusion nucleic acids encoding bothreceptors. The transfected tumor cell expresses either dual or chimericreceptors which bind SAg and GalCer independently or as fusion proteinor conjugate. Likewise, tumor cells are transfected with nucleic acidsencoding CD14, the LPS receptor, (Ferrero, E. et al., J. Immunol. 145:331-336 (1990)) a leucine rich receptor glycoprotein found on myeloidcells with a LPS binding site between amino acids 57-64. Nucleic acidsencoding CD14 are transfected into tumor cells together with nucleicacids encoding SAgs and resulting tumor cell expresses several receptorsor a single chimeric receptor with preserved consensus binding sequencescommon to each. These tumor cell transfectants are capable of bindingexogenous SAg and/or LPS and or GalCer. The resulting tumor cells withbound SAg, and/or Gal/Cer and/or LPS activate a population of T cellsand/or NKT cell to produce tumor specific effector cell which are usefulin the adoptive immunotherapy of cancer by methods in Example 1-7, 12,15, 16, 18-23. The tumor cell transfectants are also administered as avaccine or to hosts with established tumors as in Example 19-23.

Alternative splicing and utilization of cryptic splice sites generatesalternative reading frames and secretory isoforms of CD1, CD14 and SAgreceptors. Woolfson A. et al., Proc. Natl. Acad. Sci. U.S.A 91:6683-6687 (1994). These soluble receptors are immobilized on solidsurfaces such as polystyrene plates or beads and bind their respectiveligands e.g. GalCer and SAg. In this form the GalCer and SAgs activate Tcell or NKT cell to produce a population of tumor specific effector Tcell or NKT cells useful in adoptive immunotherapy of cancer by methodsgiven in Examples 7, 15, 16, 18-23).

14. SAg-Activated Tumor Specific T Cells, NKT Cells or γ/δ T CellsExpressing CD44 for Adoptive Immunotherapy

It is imperative that T cells, NKT which are stimulated in vivo or exvivo by the SAg constructs given herein are capable of trafficking andhoming effectively to tumor sites. CD44 expression on T cells after SAgstimulation, is an indicator of upregulated adhesive capacity which isrequisite for the homing of SAgs to tumor sites. T cells or NKT cells orcells transfected with nucleic acids encoding SAg receptors i.e. tumorcells or accessory cells are stimulated by SAgs in vivo or ex vivo toexpress CD44. These CD44 expressing T cells are enriched and expandedand then harvested for use in adoptive therapy of cancer by protocolsgiven in Examples 7, 15, 16, 18-23).

Transfection of cDNAs encoding soluble isoforms of CD44 into tumor cellsresults in the local release of soluble CD44 which inhibits the abilityof endogenous cell surface CD44 to bind and internalize hyaluronate andto mediate tumor cell invasion. Mice injected with tumor celltransfected with the CD44 isoform showed not tumor metastases. Suchtumor cells were shown to undergo apoptosis. These transfectantsdisplayed a marked reduction in their ability to internalize and degradehyaluronate. Therefore, CD44 function promotes tumor cell survival ininvaded tissues possible as a result of impairing their ability topenetrate the host tissue hyaluronan barrier. In the present invention,SAg-encoding nucleic acid is co-transfected or fused to nucleic acidsencoding CD44 isoforms. These transfected cells are capable of migratingto sites of metastatic tumor in tumor bearing hosts and eliciting apotent anti-tumor response. The combined apoptotic effect to the CD44isoform with the enhanced immunogenicity of the SAg produces a powerfulsynergistic anti-tumor response. The nucleic acids encoding the CD44isoform and SAg are transfected into accessory (DC)/tumor cell hybrids.In addition, to presenting tumor antigen and SAg to the immune systemand inhibiting metastases, the CD44 isoform produces apoptosis of thefusion cell which in turn is ingested by DCs resulting in enhancedimmunogenicity and a more potent tumoricidal response. These combinedtransfectants are used preferably against established tumor according toprotocols in Example 19-23.

NKT cells or T cells that do not produce CD44 after SAg stimulation doso after transfection with nucleic acids encoding CD44 or transferasessuch as N-acetylglucosaminyl transferase III or CD44 (Sheng, Y. et al.,Int. J. Cancer 73, 850-858 (1997); Nottenberg, C. et al., Proc. Natl.Acad. Sci. U.S.A 86: 8521-8525 (1992)).The latter enzyme synthesizesbisecting N-acetylglucosamine structures on asparagine linkedoligosaccharides. Glycosylation of CD44 by these transferases producesenhanced CD44 mediated adhesion to immobilized hyaluronate. SAgs areused to activate T cells which have been transfected with nucleic acidsencoding N-acetylglucosaminyltransferase III. The SAg stimulatedtransfectants display increased CD44-mediated adhesion. as well aslymphocyte homing and trafficking. Certain T cell, NKT cell or/T cellpopulations which are unable to express CD44 after SAg stimulation aretransfected with nucleic acids encoding CD44 before sensitization withSAgs. These cells express CD44 after immunization with SAgs in vivo orin vitro. These additional populations of effector T cells are useful inadoptive immunotherapy of cancer by methods given in Examples 5, 7, 15,16, 18-23.

15. Tumor Associated Antigens include

(1) Normal structures, e.g., differentiation or tissue specificantigens,

(2) Mutated normal structures

(3) Products of alternate reading frame or fusion of several genes

(4) Chimeric products resulting from cell or gene fusion

(5) Xenogeneic antigens (“xenoantigens”)

A tumor antigen (also called “tumor associated antigen) is any antigenicstructure expressed by a tumor cell. For example, tumor antigens includemutated products of various oncogenes and p53 genes that are expressedin tumor cells generally. Many tumor antigens associated with particulartypes of cancers are known. For example, tumor antigens associated withbreast, colon, and lung cancer are known and have been cloned. Commonmelanoma antigens recognized by T lymphocytes have been identified andare used as immunotherapeutic antigens for treatment of melanoma. Fivegenes encoding different melanoma antigens have been identified. Forexample, MAGE1 and 3, expressed on melanoma and other tumor cells, arerecognized by cytotoxic T lymphocytes (CTL) in the context of HLA-A1(Van der Bruggen P et al., Science 254:1643 (1991) and Gauler B et al.,J. Exp. Med. 179:921 (1994)). MART-1 identical to Melan-A (Kawakami etal., Proc. Natl. Acad. Sci. U.S.A 91:3515 (1994) and Coulie et al., J.Exp. Med. 180:35 (1994)); gp100 (Kawakami et al., Proc. Natl. Acad. Sci.U.S.A 91:6458 (1994)); and tyrosinase (Brichard et al., J. Exp. Med.178:48 (1993)) are melanocyte lineage-related antigens expressed on bothmelanoma and melanocytes. MART-1 and gp100 have been shown to berecognized by MHC-class I-restricted CTL in the context of HLA-A2, andtyrosinase in the context of HLA-A2 and HLA-A24 [Robbins et al., CancerRes. 54:3126 (1995)]. An additional list of tumor antigens useful inthis invention is given in Rosenberg, S A. Principles and Applicationsof Biologic Therapy in Cancer: Principles and Practice of OncologyDeVita, V T., Hellman, S., Rosenberg, S. A., eds, J. B. Lippincott Co.Philadelphia, Pa. 1993.

In addition tumor associated antigens are defined as including normalstructures expressed in tumor cells, mutated normal structures, normaldifferentiation- or tissue-specific structures, products of alternatereading frames of the same genetic regions, chimeric products of severalgenes that originated in a parental or in a fused, hybrid cell. Thisalso includes gene products expressed in association with MHC moleculesor other surface receptors, organelles or vesicles. Tumor cellsexpressing tumor-associated antigens are transfected in vivo or ex vivowith nucleic acids encoding a SAg alone or together with nucleic acidsencoding other products, such as those listed in Tables I and II. Theseinclude surface antigens and receptors such as the Gal epitope, GalCer,CD1, CD14 and SAg receptor. The transfected cells may be of host origin,or syngeneic, allogeneic or xenogeneic; the cells may be non-malignant.SAg-encoding nucleic acid may also be inserted into a mutated normalgene in a tumor cell, e.g., LDL receptor gene in a melanoma cell. TheLDL receptor is expressed as a fusion product of the LDL receptor gene(chromosome 19) and a fructose transferase gene on the same chromosome.This combination results from chromosome inversion which gives rise tothe fusion product probably due to recombination between the two ends ofthis chromosome. The expressed peptide epitope is therefore a nonsensesequence being read in the wrong direction. The three base pairmutations in the third open reading frame results in the expression of amutant peptide. Site directed mutagenesis can be achieved by insertionof SAg-encoding nucleic acid into the mutant gene at any feasible siteor by targeting insertion in place of the mutated base pairs. Theresultant LDL receptor displays the SAg alone or as a chimera with themutant sequence. Site directed mutagenesis by SAg-encoding nucleic acidmay also target the □-catenin gene which in melanoma shows a single C-Tmutation which results in a ser to phe substitution and the generationof the 9 amino acid mutant peptide. The SAg-encoding acid may beinserted or may substitute for any sequence in a normal non-mutatedgene, a tissue-specific or differentiation-associated gene in tumorcells, or other genes expressing their products in a tumor cell.Preferably, the. mutated gene product is immunogenic and recognized as adominant epitope by the host immune system, preferably by T cells(including tumor infiltrating lymphocytes). The mutated sequence may, incontrast, be a weak immunogen which is rendered more immunogenic whenpresented in the context of a SAg.

In the preferred embodiment, the transfected cells are tumor cells ofhost origin expressing a defined tumor associated antigen such asMART-1. If the tumor antigen is not expressed or weakly expressed on thetransfected cells, then the tumor cell is transfected with nucleic acidsencoding an immunogenic tumor antigen such as MART-1, tyrosinase orMAGE-1 in addition to SAg and other constructs described herein.

The tumor cells may be transfected in vivo by administering nucleicacids encoding SAgs and/or the other nucleic acid constructs describedabove using a site directed mutagenesis approach in vivo and methodssuch as described in Example 1, 3, 18-23. Tumor cells may also betransfected ex vivo by methods given in Example 1-3. Ex vivo transfectedtumor cells are used as vaccine or to treat established tumor by methodsand protocols in Example 18-23 They are also useful ex vivo to immunizeT cells or NKT cells to produce a population of tumor specific effectorcells adoptive immunotherapy of cancer by methods and protocols given inExamples 7, 15, 16, 18-23.

16. Immunostimulatory Sequences

Several of constructs consist of nucleic acids encoding SAg peptideswhich produce anti-tumor responses by activating host TH-1 CD4+ T cellsto proliferate and produce tumoricidal cytokines such as IL-1α, IL-1□,IL-2, IL-6, TNFα, TNF□ and IFNγ. The incorporation of theimmunostimulatory sequence into the genetic construct of SAg DNA,ensures that the T cell response is skewed to produces a predominantproliferation of TH1 cells and production of a TH1 cytokine profile.Immunostimulatory sequences (ISS) consist of DNA sequences that exhibitimmunogenicity. Briefly, plasmid DNA (pDNA) having shortimmunostimulatory DNA sequences containing a CpG dinucleotide in aparticular base context were shown to be immunogenic (Tokunaga J et al.,J. Natl. Cancer Inst. 72:955-962 (1984)). By synthesizing singlestranded nucleotides corresponding to different regions in theMycobacterium bovis genome, specific single stranded oligonucleotidesthat activate adherent splenocytes and enhanced natural killer cellactivity have been identified. In addition, single strandedoligonucleotides with CpG motifs induce B cell proliferation andsecretion of IL-6 and IFN (Krieg et al., Nature, 374:546 (1995)). Theactivation capability generally has the formula5′-Pur-Pur-C-G-Pyr-Pyr-3′. Further, human monocytes transfected withpDNA or double stranded oligonucleotides containing ISS transcribedlarge amounts of IFNγ and IL-12 (Sato et al., Science 273:352-354(1996); Zhu et al., Science 261, 209-211, (1993)) Direct gene transferwith plasmid-cationic liposome complexes resulted in lasting,generalized or tissue specific expression of the injected geneticphenotype.

In the present invention, the ISS is inserted into nucleic acidsequences of SAgs and tumor associated antigens which are used totransfect tumor cells, antigen presenting cells, accessory cellsincluding muscle cells in vitro or in vivo by methods given in Example1-3, 15, 16, 18-23. In all instances, the SAg stimulation of the T cellresponse is critical to an effective anti-tumor response of the host.The presence of the ISS ensures that the SAg nucleic acidspreferentially activate the TH1 after in vivo administration of thenucleic acids encoding SAg. SAg DNA is useful ex vivo in activating Tcells by direct transfection or by presentation via incubation withpretransfected antigen-presenting cells or tumor cells. The tumorspecific T effector cell are then useful for adoptive therapy of cancerusing protocols given in Examples 7, 15, 16, 18-23). A particularlyuseful method involves the intratumoral injection of nucleic acidsencoding SAgs. The latter is administered in naked, plasmid or liposomalform. Once tumor inflammation is initiated (generally within 15 daysafter injection), the host is given T cells or NKT cells which have beenimmunized in vitro to the tumor by tumor cells transfected with nucleicacids encoding SAg plus additional constructs given in Tables 1 and IIby methods given in Examples 7, 15 16 18-23.

17. Liposomes

Liposomes containing repeating units of the Gal epitope, GalCer, and/orSAgs are constructed and administered directly into a tumor. Theseelements are combined before incorporation into liposomes or they areadded individually in the preparative procedure. Methods for preparationof these liposomes are given in Examples 5. These liposomes arepreferentially delivered parenterally or directly into the tumor. Theadministration of SAgs in this manner provides a high localconcentration of SAg to stimulate an anti-tumor response. Theseliposomes are also useful ex vivo by activating a T cell or NK T cellpopulation which is then harvested and used for adoptive immunotherapyas described in protocols in Examples 5, 7, 15-17, 18-23).

18. Tumor Cells that Induce Cellulitis

Transfection with microbial nucleic acids that encode tissue spreadingfactor (hyaluronidase), erythrogenic toxins, enterotoxins; capsularpolysaccharides from S. aureus and Streptococcus pyogenes, S. aureus andS. pyogenes have potent tissue invasive properties. Specifically,Staphylococcus and Streptococcus are capable of invading tissues bysecreting several enzymes which lyse ground substance such asmucopolysaccharide, hyaluronic acid, or chondroitin sulfate, createlocal thrombosis, and initiate inflammation and edema. These enzymesconsist of hyaluronidase, streptokinase, streptodornase, erythrogenictoxins as well as various enterotoxins (Example 3). In the presentinvention, the nucleic acid sequences encoding these potent enzymes aretransfected into tumor cells, either in vitro or in vivo (Examples1-3,6, 15, 16, 18-23). In vivo, the transfected tumor cells migrate to sitesof existing metastases. The transfected tumor cells secrete the enzymeswhich hydrolyze the tumor ground substance and neovasculature and toxinsto induce inflammation and an immune response in tumor tissue. Tumorswhich are encased in nests of connective tissue are eliminated by thisprocess. The resulting increase in local vascular permeability inducedby the combined effect of enzymes and toxins produces intenseinflammation at tumor sites. If their administration is timed to thepeak of tumor inflammation, liposomes as described herein andchemotherapy are sequestered and concentrated in the inflamed tumor bedproducing an augmentation of the tumoricidal response.

A relatively low number of transfected tumor cells with the completemicrobial enzymatic and toxin genetic construct would be required toinduce a tumoricidal effect. The population of transfectants would thenproceed to secrete these microbial enzymes locally. In addition, nucleicacid encoding these enzymes are derived from a strain of Staphylococcusor Streptococcus such as Staphylococcus epidermidis or Streptococcusbovis of low or intermediate virulence.

Tumor cells are cotransfected with glycosyltransferases or treated withglycosyltransferase-inducing agents resulting in the expression of theGal epitope and reduction in the survival time of tumor cells Forexample, the nucleic acids encoding the glycosyltransferase fromSphingomonas paucimobilis or Agelas mauritianus produce GalCer aretransfected into tumor cells to induce the surface expression of GalCeror Gal. The tumor cells then express and/or secrete microbial agentssuch as SAgs, hyaluronidase and erythrogenic toxins that hydrolyze theground substance of the tumor. By also displaying SAgs and -Gal orGal/Cer epitopes which activate NKT cells, T cells, and Gal specificantibodies the transfected tumor cells induce profound tumoricidalactivity. These transfected tumor cells are used to activate apopulation of T cells to become tumor specific effector cells which areemployed for the adoptive immunotherapy of cancer. See Examples 1, 2,4-5, 7, 15, 16, 18-23.

For in vivo transfection of tumor cells, the microbial genetic nucleicacids are targeted to tumor cells as described herein (See p. 12“Transfection”, Examples 1-3, 6, 19). Once localized in tumor sites invivo, the tumor cell is capable of hydrolyzing surrounding stroma and,initiating thrombosis, inflammation, and increased tissue permeability.Additional microbial nucleic acid encoding proteinases, lysoproteinases,tissue spreading factors, α and □ hemolysins and toxins are alsotransfected into tumor cells and used in accordance with this invention.

Micrometastatic disease in cancer patients is of great concern as itoften goes undetected and is refractory to chemotherapeutic agents.Documented metastases in breast cancer patients is associated with apoor prognosis. The present invention contemplates that the metastaticproperties of tumor cells coupled with the potent inflammatoryproperties of the microbial products are useful in tracking andeliminating micrometastatic disease in tumor bearing patients. Tumorcells are transfected with nucleic acid encoding polypeptides involvedin metastasis. These include but are not limited to peptides thatupregulate the adhesive properties of CD44 (e.g., glycosyltransferases),the c-erbB-1 encoded EGF receptor which is associated with enhancedmetastases in breast carcinomas or c-erbB-2/neu encoding the p185receptor associated with poor prognosis in breast and ovariancarcinomas. These cells with metastatic activity are programmed totraffic, home and colonize specific sites of existing metastases thetumor bearing host. Hence they have the unique property of charting themicrometastatic sites of the tumor. These tumor cells are cotransfectedwith microbial nucleic acids encoding the hyaluronidase, erythrogenictoxin and enterotoxins as well as the Gal. Hence, as they colonizemetastatic sites, these transfectants induce a potent inflammatory andimmune response. This ensures their own destruction together with thesurrounding untransfected micrometastatic tumor cell population andneovasculature and stroma. Methods of preparation, administration andassessment of these transfectants in tumor bearing hosts are in Example1-3, 18-23.

The tumor cells are also transfected with the above microbial genes on aDNA template with a tissue specific promoter in order to target theactivity of these transfected tumor cells to the vital organs (and sitestherein) affected by the existing metastatic tumor. For breast cancer,this would be lung, liver or brain. These organ-specific promotersensure that the expression of the microbial products would occur in theorgan(s) targeted by the tissue specific promoter The same tumor cellsare also provided with inducible promoter sequences which control thelevel of receptor transcription and expression. Inducible promoterssuitable for use in mammalian cells include the MMTV-LTR under thecontrol of steroid hormones and the metallothionein promoter under thecontrol of heavy metal ions. In this case, the microbial nucleic acidsare linked to steroid inducible gene sequences. Transcription istriggered when these cells are exposed to a threshold level of steroids.Hence, two to three days after administration to the host, when theabove transfectants have colonized tumor metastatic sites, a bolus ofcorticosteroid is administered which initiates transcription of themicrobial enzymes and toxins by the tumor cell transfectants and theirsecretion. In this fashion, the transfected tumor cells express andsecrete their inflammatory products in metastatic tumor sites resultingin the elimination of metastatic disease.

19. Tumor Cells as Mimics of Virulent Bacteria: Transfection withNucleic Acid Encoding Bacterial Invasins, Virulence Factors, and Enzymesthat Degrade Extracellular Matrix

Tumor cells with a metastatic phenotype are transfected with nucleicacids encoding proteins with the capacity to invade and adhere toinflammatory cells such as macrophages (adhesins and virulence factors).These genes are inducible and controlled by operons.

SAg-encoding nucleic acid is fused in frame to nucleic acid encodingoncogenes involved in tumorigenesis and metastasis. Examples of suchgenes, in addition to erb/neu, erb, erbB2 and EGF (epidermal growthfactor receptor) discussed above, include ras and mutated ras, erk, andmtal, 182mts1, nm23 (See Table 9.5, p181 of Franks L. M. et al.,Cellular and Molecular Biology of Cancer, Oxford University Press,Oxford UK, (1997) which is incorporated by reference), as well as thelaminin-integrin and the cadherin family. These genes are particularlyuseful because they are overexpressed in tumor cells displaying ametastatic phenotype.

Invasins

SAg-encoding nucleic acid is fused in frame or cotransfected into tumorcells with nucleic acids encoding bacterial invasins and hyaluronidases.The invasin imparts leukocyte like activity to bacteria is transfectedinto tumor cells which allows the tumor cells to penetrate tissues.These are exemplified by Yersinia pseudotuberculosis invasin andhyaluronidase (including its various isotypes) and also known as tissuespreading factors. The invasin gene exemplified in Y. pseudotuberculosisencodes a protein located in the outer membrane of the bacterium calledinvasin (Inv) and the gene is known as inv. The DNA region of the invgene contains a open reading frame 2964 bases. This protein binds to thehost cell surface by means of the C-terminal 192 residue region.Mutation by insertion of a transposon or elimination of the inv genegreatly impairs the ability of the bacterium to penetrate tissues(Schaecter M et al., Genetics of Bacteria edited by Baer G M et al., inMechanisms of Microbial Disease Williams and Wilkins Baltimore (1993)).

The host membrane receptors for invasin belong to the integrinsuperfamily with a particular affinity for VLA-3, 4, 5, 6. Invasin alsobind to T cell α₄□₁ which is involved in lymphocyte homing or traffic.Once bound to a phagocyte, phagocytosis is triggered and the bacteriumis taken up. Nucleic acids encoding Inv are transfected into tumor cellswhich confers upon the tumor cell a phagocytosis triggering signal forhost macrophages.

E. coli genes of the P pili or pap operon encoding adhesin proteins havebeen isolated from chromosomes and plasmids. The gene cluster is linkedto genes for other virulence determinants such as the KI capsularpolysaccharide and hemolysin. The receptor for the pili is theGal(1-4)Gal moiety of the P blood group antigen. Examples of host cellreceptors for bacterial adhesins is given in Table 7.2 of Patrick andLarkin. Pilin genes in N. meningitidis encode proteins is which thefimbriae are the N-methylphenylalanine pili. An extensive region ofamino acid homology at the N-terminal end is common to a wide range ofbacterial genera including Pseudomonas aeruginosa, N. gonorrhoeae, N.menigitidis, Moraxella bovis and Bacteroides nodosus. This N-terminalregion is highly hydrophobic which is in contrast to the fimbriae of theEnterobacteriaceae which either have a hydrophobic region at theC-terminal end or lack a hydrophobic region altogether. Of interest isthe presence of a site on SAgs which resembles the third Ig-likedisulfide-bridged loop of VCAM-1 and a conserved sequence is presentwithin the same subregion of the fifth Ig-like VCAM-1 loop. The onlyknown receptor for the VCAM-1 is VLA-4, an adhesion molecule expressedprimarily by activated T and B cells. A survey of target cellsusceptibility to SEC dependent lysis shows a correlation between VLA-4expression and susceptibility to lysis.

Hyaluronidases and Proteases

Bacteroides species produce hyaluronidase, heparanase, and chondroitinsulfatase enzymes. C. perfringens m toxin is a hyaluronidase enzyme andBacteroides and C. perfringens produce elastase and collagenase enzymewhile Porphyromonas gingivalis has a cell associated collagenase.Streptococcus pyogenes produces hyaluronidase enzymes which depolymerizetheir own capsules. Neuraminidases and endoglycosidases, lipases,nucleases and proteases produced by a wide variety of bacteria are alsouseftil in this invention as capable of promoting tissue necrosis intumor masses and/or tumor nests.

The staphylococcal invasive genome is predominantly chromosomal and thenucleic acid segments encoding the major invasive enzyme systems,permeability factors, and toxins have been isolated, cloned, andsequenced. For example, the nucleic acid sequence encoding ahyaluronidase from group A Streptococcus strain 10403 is describedelsewhere (Hynes et al., Infect. Immun. 63:3015-3020 (1995)). Tumorcells transfected with nucleic acids encoding microbial invasive andinflammatory substances are preferentially used in vivo where they areprogrammed to traffic to metastatic sites and/or organs primarilyinfiltrated by the tumor. Once situated in tumor, they commencesecretion of their inflammatory enzymes and toxins. Protocols for theirpreparation, use, and assessment are given in Examples 1-3, 18-23.

Consolidation of Bacterial Genes

The microbial nucleic acids encoding hyaluronidase, erythrogenic toxinsproteases, coagulases and enterotoxins are consolidated into a chimericconstruct or plasmid and transfected into tumor cells which thencommence secretion of the spreading factors, pro-inflammatory andpermeability inducing agents. For example, a single construct ormultiple constructs contains the nucleic acid encoding polypeptidesincluding, without limitation, enterotoxin B, hyaluronidase,streptokinase, coagulase, Staphylococcal protease and erythrogenictoxins.

Tumor cells transfected with the above microbial genes are prepared asin Example 1-3 and are used in the treatment of established andmetastatic tumor or as a preventative vaccine as described in Examples15-23.

20. Combined Expression of Different Stimulatory Molecules byCo-Transfection of Tumor Cells or Fusion of Singly Transfected Cells

Tumor cells that express two different types of exogenous molecules areproduced by either cotransfection of the same cells with (a)SAg-encoding nucleic acid and (b) nucleic acid encoding a toxins orautolysin, or by fusion of tumor cells that have been singly transfectedwith (a) with tumor cells transfected by (b) Tumor cells are providedwhich have the dual capacity to colonize metastatic tumor sites in vivoand induce inflammation. Once situated in sites of tumor metastasis, thetumor cells behave like a necrotizing bacterium or leukocyte. Forexample, tumor cell are transfected with nucleic acids encodingbacterial invasins to promote adhesion, “tissue spreading factor” orhyaluronidase to hydrolyze the ground substance, coagulase to inducelocal thrombosis and streptokinase and streptodornase. In addition,tumor cell are provided with nucleic acids encoding bacterial toxinswhich bind and produce autolysis and cytotoxicity for surrounding tissueand tumor cells. The tumor cells are also cotransfected with additionalnucleic acids encoding SAgs. The toxin genes useful herein are amplifiedby providing two copies tandemly duplicated on a chromosome and linkedto an amplified oncogene. Situated in tumor tissue, these transfectedtumor cells release enterotoxins as well as inflammatory enzymes,immunogenic capsular lipoproteins, cell wall LPS's and cytolysins. Thisevokes a potent T cell and inflammatory response in tumor tissue. Theseinflammatory genes are inducible at the level of the operon or in someinstances bacteriophage which controls their activation. Transfectedtumor cells are transfected with microbial nucleic acids given aboveeither in vitro or in vivo at tumor sites as in Example 1-3, 5, 16-23and p.11 under “transfection”.

The S. aureus a toxin forms pores or transmembrane channels in a widerange of host cells. It is released from the bacteria during exponentialgrowth and has a molecular mass of 33 kDa. Expression of the geneencoding the a toxin, hly, is under the control of the agr gene whichcoordinately controls the expression of a number of extracellularproteins, including exfoliatin toxin, toxic shock syndrome toxin, α, □,and δ toxins, enterotoxin B, lipases and nucleases. The □ toxin is aphospholipase which attacks a sphingomyelin in the cell membranes. Thephage encoding the toxin is hlb. Exfoliatin toxin A is encoded by achromosomally located gene eta and the gene for toxin B is etb. The etagene is by the agr gene regulator which is a member of thehistidine-protein kinase response regulator superfamily. (Patrick S etal., Immunological and Molecular Aspects of Bacterial Virulence, JohnWiley and Sons New York, N.Y. 1995)

SEB binds to glycosphingolipids on cell membranes. The gangliosidebinding site on SEB is overexpressed, or a myristoylation site or GPIbinding site is integrated into its structure so that it is bound to thesurface of the tumor cell membrane and not secreted. The SEB willpreferentially bind to tumor cell expressing ganglioside tumorassociated antigens and will augment the immunogenicity of theseantigens.

S. aureus produces a bifunctional protein autolysin of 110-kDa,(HlyA)via the atl gene that has an N-acetylmuramoyl-L-alanine amidase domainand an endo-□-N-acetylglucosaminidase domain. It undergoes proteolyticprocessing to generate two extracellular enzymes that are secreted. Thespecific secretion proteins HlyB and HlyD are 80 kDa and 54kDarespectively. The process is directed by the hlyB and hlyD genes whichare contiguous and co-expressed with the hylC and hylA genes that arerequired for the synthesis of protoxin and the acyl carrierprotein-dependent fatty acylation that matures it to cytolyticallyactive toxin. Hemolysin is secreted as the mature acylated form of thehlyA gene product proHlyA following the covalent attachment of a fattyacid moiety in a cytoplasmic mechanism directed by the dimeric HlyCactivator, a putative acyl transferase and dependent upon the acylcarrier protein. This specific and novel HlyC-directed fatty acylationis required to target the hemolysin toxin to mammalian cell membranesprior to forming cation-selective pores and disrupting the host cell.

Bacteria such as E. coli, Bordetella pertussis, Pasteurella haemolytica,Proteus vulgaris and P. mirabilis produce genetically related toxins.Their activity is dependent on the presence of calcium ions.Characteristically, they have regions of 10 to 47 repeats within theamino acid sequence and termed repeats in toxin or RTX gene family. Therepeat sequence contains the following nine amino acids (SEQ ID) NO:34);leucine-X-glycine-glycine-X-glycine-asparagine-aspartic acid-X where Xis a variable amino acid. These repeats are required for hemolyticactivity. A large hydrophobic region of the hemolysin separate from therepeats, is also essential for activity and may be involved in theinteraction with the host cell membrane. The hemolysin A of E. coliapparently form pores on the target cell membrane. This requires a 20kDa product of another gene HlyC before it becomes actively hemolytic.In E. coli, the operon for the production of the hemolysin contains fourgenes hlyA which codes for the structural hemolysin and hlyc which isrequired for activation of the HlyA. The other two genes hlyB and hlyDare involved in the transport of HlyA to the extracellular environment.Pasteurella haemolytica leukotoxin and Bordetella pertussis adenylatecyclase hemolysin have similar C-terminal sequence and associated genesanalogous to those in the hly operon. (Koronakis V et al., Secretion ofHemolysin and other Proteins out of the Gram-Negative Bacterial Cell, inGhuysen J M et al., ed, Bacterial Cell Wall, Elsevier, Amsterdam(1994)).

The Shiga toxin of Shigella dysenteriae and Shiga-like toxins of E. coli(Verotoxins) are a family of related toxins which have similar aminoacid sequences and biological activities. The A subunit of Shiga toxinhas a molecular mass of 31 kDa which associates with five to the 7 kDa Bsubunits. The A subunits is proteolytically cleaved into A1 and A2. Itis the A1 fragment which is biologically active. The host cell receptorfor Shiga toxin is the glycolipid Gal(α1-4)Gal(□1-4) GlcCeramide(globotriosylceramide; Gb3) and for Shiga-like toxin I (SLTI) and SLTIIof E. coli is Gal(α1-3)GalCeramide (Galabiosylceramide). The bindingspecificity is dependent on both sugars residues and the lipid moiety.The Shiga toxin is known to inhibit protein synthesis. It is a RNAN-glycosidase enzyme whose site of action is the 60S ribosomal subunit.The toxins remove an adenine base from position 4324 on theaminoacyl-transfer RNA binding site of 28S ribosomal RNA hencepreventing peptide length elongation. The effect on protein synthesis issimilar to that of diphtheria toxin and Pseudomonas aeruginosa exotoxinA. The SLTI and II toxins of E. coli and encoded by lysogenic phage. Itsexpression is controlled by iron concentration in the growth medium byway of the fur gene and iron box repressor protein binding site.Clostridia difficile toxins A and B also bind to anomeric galactoseepitopes on cell membranes and induce membrane associated enzymes andinhibit G protein activation which results in cell death. Tumor cellstransfected with a galactosyltransferase genes to produce the α-Galepitope are susceptible to lysis by both the Shiga-like toxins and C.difficile toxin. The expression of the α-Gal epitope is enabled by thetransfection of nucleic acids encoding -Gal transferase into tumorcells.

Listeria monocytogenes produces a hemolysin. listerolysin O (LLO), amember of the thiol-activated family of cytolysins. LLO is encoded bythe gene hyl (also designated hylA and lisA). Listerolysin O toxin is apore forming toxin which degrades the membrane of its phagocytic vacuoleallowing the bacterium to escape into the host cytoplasm. This genecloned into Bacillus subtilis enables the bacterium to grow rapidlyintracellularly in the cytoplasm of a macrophage-like cell line afterdisrupting the phagosomal cell membrane. Tumor cells are transfectedwith the above microbial nucleic acids as in Example 1-3. Thesetransfectants are useful in vivo against established tumor andmicrometastatic disease (Examples 5, 15, 16, 18-23).

21. Augmentation of Tumor Cell Immunogenicity by Bacterial Products:Transfection with Genes Encoding Bacterial Antigens or Receptors forBacterial Products

Tumor cell are provided with augmented antigenicity by expressingfundamental patterns that are recognized by fundamental recognitionunits of the innate immune response. Examples are LPS's of gram negativeorganisms, SAgs and peptidoglycans of gram positive organisms, fungal□-glucans, bacterial glycosylceramides, and mycobacterial lipoarabinans.Numerous infectious agents with these structures cause potent immunereactions e.g. streptococcal cellulitis induced by S. pyogenes, E. coliinduced sepsis and meningococcal meningitis induced by Neisseriameningitidis (SEQ ID NOS:35-36)..

The T cell system is far more adept at responding to innate patternrecognition units than to tumor associated antigens. In the presentinvention, tumor cells are transfected with nucleic acids encodingmolecules or biosynthetic enzymes that result in structures which mimicthe major immunogenic structures of bacterial antigens. This enables thetumor cells to be recognized more effectively by the T cell system. Inaddition, tumor cells are provided with receptors for bacterial antigenssuch as SAgs, LPS's (CD14), and glycosylceramides (CD1). Genes encodingbacterial antigens which produce potent immune responses are transfectedinto tumor cells to include bacterial membrane and cell wallconstituents such as LPS's, peptidoglycans, glycosylceramides,lipoproteins, lipoarabinans and capsular polysaccharides. In addition,nucleic acids encoding the staphylococcal SAgs induce potent T celllymphoproliferation and TH-1 cytokine production while LPS's are knownto have a bystander effect on T cell proliferation The two agentssynergize in their capacity to induce lethal endotoxic shock in animals.The present invention contemplates that the optimal approach is topresent the bacterial immunogen structure (for example streptococcalcapsular polysaccharide) sequentially or concomitantly with a bacterialmitogenic signal (SAg). Under certain conditions, these genes areco-transfected with various bacterial invasins, toxins, autolysins andinflammatory enzymes which together with the colonizing properties oftumor metastasis genes produce a tumor cell capable of migrating tometastatic sites where it induces necrotizing cellulitis. Such genes arepreferably placed under the control of inducible promoters as describedherein.

These transfectants are prepared by methods in Example 1-3. They areuseful against established tumors or metastatic tumor in vivo as inExample 15, 16, 18-23.

21b.Combining Expression of SAg Nucleic Acids with Nucleic AcidsEncoding Enzymes that Drive the Synthesis of Bacterial LPS,Galactosylceramide or Capsular Polysaccharide

In general, this is accomplished by co-transfection of nucleic acidseach encoding one of the above products or by transfection with a fusionnucleic acid that encodes the combination.

SAg-encoding nucleic acid is fused in frame or cotransfected into tumorcells or accessory cells with nucleic acids encoding bacterial LPS's,peptidoglycans, and galactosylceramides. The preferred end products aresynthesized in E. coli and N. meningitides (LPS's), Staphylococcus andStreptococcus (peptidoglycans); Sphingomonas paucimobilis(glycosylceramides).

The synthetic genome or cluster of genes for biosynthesis of theseproducts is incorporated as a whole to include multiple and specificenzymatic transferases and trafficking proteins required for thestepwise synthesis of each of these products. Gene clusters arenecessary to provide the requisite transferases for synthesis of theselarge molecules. For example the genes required for the biosynthesis oftype 1 capsular polysaccharide of S. aureus are localized to a 14.6-kbregion. Sequencing analysis of the 14.6-kb fragment revealed 13 openreading frames (ORFs). Ten genes are involved in capsule biosynthesis.CapG aligned well with consensus sequence of a family ofacetyltransferases from various prokaryotic organisms suggesting thatCapG may be an acetyltransferase. The structural requirements forendotoxic activity of LPS's are as follows. (1) a □(1-6)-linkedD-glucosamine disaccharide backbone; (2) biphosphorylation at positions1 and 4′ of the disaccharide backbone; (3) a suitable number of3-acyloxyacyl groups per disaccharide unit; and (4) acyl groups ofa-suitable length as indicated by Kumazawa et al., and Nakatsuka et al.Transfection with nucleic acid encoding LPS's would require thepreservation of the biphosphorylation and the acyl groups between 14 and23 to maintain optimal activity. Derivatives may contain amonosaccharide group in place of the disaccharide group.

LPS Structure

LPS consists of an outer region which is composed of polymerized di- andpentasaccharide repeating units whose compositions vary within a speciesor strain. The inner region is generally conserved within a singlegenus, and consists of a core oligosaccharide linked by the sugar2-keto-3-deoxy-D-amino-octonate (KDO to a disaccharide backbone withattached long chain fatty acids, the lipid A. This component isresponsible for much of the biological activity of the molecule.Components conferring the greatest biological and immunomodulatoryactivity are now known to be a glucosamine disaccharide, a bisphosphorylated lipid A and acyloxyacyl groups on the fatty acid chain.The loss of only one of these components, for example, a phosphate groupreduces the activity of the molecule. LPS's from different genera ofbacteria vary in their immunomodulating activity and studies of thestructure have shown very subtle differences. For example, Bacteroidesspp. is apparently less active in endotoxin activity than LPS fromenteric bacteria. This was initially thought to be related to amodification of the of KDO in the core region with an added phosphategroup. Other differences in the LPS were found when the fatty acids fromE. coli and Bacteroides were compared. E. coli has six fatty acid chainsor acyl groups per diglucosamine backbone each with a chain length of12-14 carbon atoms. Included in the acyl groups is3-hydroxytetradecanoic acid (3-OH-C14:0)which is absent in theBacteroides strains. In contrast, Bacteroides has 4-5 fatty acids ofchain length 15-17 carbons per diglucosamine and has branched 3-hydroxyfatty acids. Studies of synthetic lipids have confirmed that reducedbiological activity relates to fewer fatty acids chains.

A common feature of LPS's from various species is that they areamphiphiles, with both a hydrophobic part capable of dissolving in lipidmembranes and a hydrophilic part which remains in the water phase.Therefore, the first step of molecular interaction is one between theamphiphilic molecule and the mammalian cell surface either by ionicbinding, hydrogen bonding or hydrophobic interaction. The bacterialmolecule may be inserted into the mammalian membrane by its hydrophobicmoiety or attached to membrane receptors with the hydrophilic moiety, orthrough charge effects or via binding to host glycoproteins andglycolipids resulting in signal transduction. Most of theimmunomodulating activity of these bacterial molecules is indirect andstems from the release of host mediators. Cytokines such as IL-1, tumornecrosis factor, and IL-6 are involved. The LPS binding protein attachesto gram-negative bacteria or free LPS and mediates the attachment tomacrophage membrane receptor known as CD14. The recognition of the CD14only recognizes LPB when it is bound to LPS. The LPS-LPB complex maydirectly trigger TNF release or hold the complex at the cell surface sothat other hosts cell surface molecules trigger TNF release. LPBs alsoact as opsonins. Another area where sugar residues play an importantrole is in cell surface glycoprotein interactions which involveprotein-carbohydrate recognition. In the recirculation of andrecruitment of leukocytes in the body, the carbohydrate-recognizingprotein domains of glycoproteins of one cells bind specifically to theoligosaccharides of glycoconjugates on another type of cell. Theserecognition events control the movement of bloodbome lymphocytes intolymphoid organs. Specific recognition occurs between lymphocytes andspecialized cells in the wall of blood vessels known as high endothelialvenules.

Genes Encoding Lipid A Biosynthesis

LPS is generally synthesized as two separate components, the lipidA/core and the O polysaccharide, which are then ligated to give thecomplete LPS molecule. Three genes encode enzymes that catalyze thesteps of lipid A synthesis (lpxA, lpxD and lpx B for steps 1,3 and 5)and fabz and envA. More specifically, the enzymes that catalyze thesynthesis of lipid A are thought to act in the following sequence(indicating the genes): lpx A, lpx C, lpx D, lpxB. The reactionscatalyzed by the products of these genes are given in Table 1 ofSchnaitman C A et al., Microbiol. Rev. 57: 655-682 (1993).

Blocks of Genes Involved in LPS Biosynthesis

Blocks of genes involving LPS synthesis have been sequenced andanalyzed. The lipid A biosynthetic pathway has been elucidated. Four ofthe genes in this pathway have now been identified. Three of them arelocated in a complete operon which also contains genes involved in DNAand phospholipid synthesis. Genes involved in synthesis of the LPS lipidA core are given in Tables 1 and 2 and their activity at various pointsin the biosynthetic pathway are given in FIG. 1 of Schnaitman C A etal., Microbiological Reviews 57: 655-682 (1993). which is incorporatedby reference. Therefore, it is likely that LPS biosynthetic enzymes areorganized into clusters on the inner surface of the cytoplasmic membranearound a few key membrane proteins.

A cluster of assembly genes produced by various bacteria encode LPS withhomologous structures. These genes have been transfected into E. coliand they induce identifiable LPS's. There are also smooth and roughLPS's which have a hierarchy of potency in terms of procoagulantactivity and activation of TNF. Mutants produced which synthesizedprogressively less polysaccharide attached to the lipid A moiety. Thepresence of long chain polysaccharides attached to the lipid moietydecreased the ability to activate TNF. Rough bacteria were moreeffective than smooth bacteria in inducing TNF production. Fatty acidsof various chain lengths can be produced including those that resemblemonogalactosylceramides. Transferases for biosynthesis of galactan theLPS structure of the O antigen from Klebsiella pneumoniae have beenidentified as well as genes controlling the O antigen chain length.

The genes for LPS and glycosylceramide assembly also involve multipletransferases. The transfection of tumor cells involves 10 genes encodinga particular stretch of the bacterial genome. In E. coli, the14-kilobase pair chromosomal region located between waaC (formerly rfaC)and waaA (kdtA) contains genes encoding enzymes required for thesynthesis and of the type R2 core oligosaccharide in the lumen of theendoplasmic reticulum. This occurs in a stepwise fashion. The geneencoding the Haemophilus influenzae type B outer membrane proteinfunctions as a porin and is useful in protective immunity has beencloned as a 10-kilobase Hib DNA insert and expressed in E. coli. Thebiosynthesis of LPS's involves genes encoding the key transferasesincluding rfaI. The N. meningitides highly conserved surface proteinconferring protection is encoded by a ORF of 525 nucleotides.

Genes Encoding Enzymes the Catalyze Core Biosynthesis

The rfa cluster includes the genes for all transferases for assembly ofcore. It includes three operons consists of at least 17 genes. Themajority of known genes whose functions are involved exclusively in LPScore biosynthesis are located in the rfa cluster [Pradel E et al., J.Bacteriology 174: 4736-4745 (1992)]. It includes three operons. However,there are also genes such as kdsA and rfaE located outside the rfacluster which are involved in biosynthesis of sugars unique to the coreor exert direct effect on core structure. These clusters appear to haveoriginated by the exchange of blocks of genes among ancestral organisms.There are few which code for the integral membrane proteins. Thepromoter for the rfa genes has been identified. Mutations have beenidentified known a rough mutants traced to three loci namely rfa, rfband rfc.

The region of the E. coli chromosome encoding enzymes responsible forthe synthesis of the LPS core has been cloned. This region formerlyknown as the rfa locus comprises 18 kb of DNA between the markers tdhand rpmBG. The genes are arranged in three different operons and thegenetic organization of this locus seems to be identical in E. coli K-12and S typhimurium.

Linkage of LPS Transcription and Toxin Secretion

In E. coli and Salmonella, a link has been found between toxin secretionand the gene regulating LPS transcription. Toxin secretion is regulatedby gene expression within the hlyCABD operon. A recently identifiedactivator of hlyCABD gene expression is the 128-kDa product of the rfaH(sfrB) gene which positively regulates transcript initiation andpossibly termination in the operons encoding synthesis of LPS of E. coliand Samonella. The discovery of a role in hlyCABD expression for the LPS(rfa) operon transcriptional activator rfaH is consistent with the roleof LPS in influencing both the secretion and toxic activity of thetoxin.

Genes Encoding Enzymes that Synthesize Polysaccharide Capsule andMembrane Proteins

Genes for the biosynthesis of a polysaccharide capsule are induced inSphingomonas by overlapping DNA segments which span about 50kbp restoredthe synthesis of sphingan. The polysaccharide components of LPS from B.Pertussis, H. influenzae and Bacteroides spp. will activate B-cells. Thepolysaccharide of Bacteroides activates B cells indirectly by firsttriggering the macrophage whereas the lipid A moiety triggers the Bcells directly. Therefore different parts of the same molecule interactwith different types of host cells. There is also evidence thatimmunopotentiating activity of a glycopeptide produced by mycobacteriais dependent on the saccharide residues of the molecule.

The capsular polysaccharide of the Streptococcus is extremelyimmunogenic, consisting of glycan strands composed of regularlyalternating N-acetylglucosamine and N-acetylmuramic acid residues joinedthrough □-1,4 glycosidic linkages and attached to crosslinked peptidesby amide bonds. The capsule of strain M is composed oftaurine-2-acetamido-2-deoxyfucose and 2acetamido-2-deoxy-D-galacturonicacid. The gene for this structure called cap-1 has been cloned and isused to transfect tumor cells. The nucleic acid sequences appear in Linet al., J. Bacteriol. 176, 7005-7016 (1994).

A new 24-kDa group A streptococcal membrane protein known asstreptococcal protective antigen (Spa) has been identified and isdistinct from the surface M protein which evokes protective opsonizingantibodies. The Spa-encoding gene has been cloned and consists of a636-bp 5′ fragment. (Dale, J B et al., J. Clin. Invest. 103: 1261-67(1999)).

The present invention contemplates the use for cancer treatment of theseand other bacterial antigens from staphylococci, streptococci, E. coli,N. mengitides, and other genera which antigens evoke an immune responsein mammals. In the preferred approach, a nucleic acid encoding such anantigenic structure is transfected and expressed in tumor cells. Methodsof preparation, use and assessment of these therapeutic constructs intumor bearing hosts are in Example 1, 2, 18-23.

SAg nucleic acids are fused in frame or cotransfected into tumor cellsor accessory cells with nucleic acids encoding key transferases (geneclusters) and glycosylation sites encoding capsular membrane fromStreptococcus or Neisseria menigitidis lipoprotein-LPS-phospholipid andcell wall peptidoglycans, i.e., N-acetylglucosamine (NAG) andN-acetylmuramic acid (NAM).

SAg DNA is fused in frame to DNA encoding a highly conserved outermembrane surface protein of N. meningitides known as Nspa. The Nspa genehas been cloned (Martin, D. et al., J. Exp. Med. 185: 1173-1183 (1997)).The LPS produced would be of weak to intermediate strength such as thatproduced by Listeria or Legionella.

Borrelia burgdorfi is the causative agent of Lyme disease. The osp genesare located at a single genetic locus on a 49kb double-stranded DNAlinear plasmid where they are organized as an operon ospAB. The aminoacid sequences of OspA and OspB show a high degree of similarity andresemble prokaryotic lipoproteins. Nucleic acids encoding the ospA andospB lipoproteins are cotransfected into tumor cells together with SAgs.

Genes Encoding Membrane Glycosylceramide Biosynthesis

Nucleic acids encoding the synthesis of the GalCer from Sphingomonaspaucimobilis are transfected into tumor cells, resulting in thesynthesis of GalCer by the tumor cell. (Kawahara K et al., FEBS Letters292: 107-110, (1991) Yamazaki M et al., J. Bacteriology 178: 2676-2687(1996) Natori T et al., Tetrahedron Letters 34: 5591-5592 (1993)Costantino V et al., Liebigs Ann. Chem. 96: 1471-1475 (1995)). Nucleicacids encoding enzymes responsible for synthesis of Neiserriameningitides LPS are transfected into tumor cells, resulting in thesynthesis of LPS by the tumor cell (Steeghs L et al., Gene 190: 263-270(1997)). These nucleic acids encoding key transferases are fused tonucleic acids encoding amplified oncogenes or transcription factors suchas Bcl-2, c-myc, K ras, bcr, c-abl or NF-κB.

Genes Involved in Mycobacterial Cell Wall Biosynthesis

SAg-encoding nucleic acid is fused in frame or cotransfected into tumorcell with nucleic acids encoding the key enzymes involved in thebiosynthesis of mycobacterial cell wall mycolic acid,phosphatidylinositol mannosides and lipoarabinans. A high affinityinteraction of CD1b molecules with the acyl side chains of known T cellantigens such as lipoarabinomannan, phosphatidylinositol mannoside andmonomycolate has been demonstrated. Hence the nucleic acid encoding theCD1 receptor are cotransfected into tumor cells together withSAg-encoding nucleic acid and nucleic acids encoding the multifunctionalfatty acid and mycocerosic acid synthases involved in the biosynthesisof mycolic acid and methyl-branched fatty acids.

The multifunctional genes for mycocerosic acid synthase involved in thebiosynthesis of these molecules have been isolated. In addition to theusual fatty acids found in membrane lipids, mycobacteria have a widevariety of very long-chain saturated (C18-C32) and monounsaturated (upto C26) n-fatty acids. The occurrence of α-alkyl □-hydroxy very longchain fatty acids i.e., mycolic acid is a hallmark of mycobacteria andrelated species. Mycobacterial mycolic acids are the largest (C70-C90)with the largest -branch (C20-C25). The main chain contains one or twodouble bonds, cyclopropane rings, epoxy groups, methoxy groups, ketogroups or methyl branches. Such acid are the major components of thecell wall, occurring mostly esterified in clusters of four on theterminal hexa-arabinofuranosyl units of the major cell wallpolysaccharides called arabinogalactans. They are also found esterifiedto the 6 and 6′ positions of trehalose to form “cord factor”. Smallamounts of mycolate are also found esterified to glycerol or sugars suchas trehalose, glucose and fructose depending on the sugars present inthe culture medium. Mycobacterium also contains several methyl-branchedfatty acids. These include 10-methyl C18 fatty acid (tuberculostearicacid found esterified in phosphatidyl inositide mannosides),2,4-dimethyl C14 acid and mono-, di- and trimethyl-branched C14 to C25fatty acids found in trehalose-containing lipo-oligosaccharides,trimethyl unsaturated C27 acid (phthienoic acid), tetra-methyl-branchedC28-C32 fatty acids (mycocerosic acids) and shorter homologues found inphenolic glycolipids and phthiocerol esters and multiple-methyl-branchedphthilceranic acids such as heptamethyl-branched C37 acid and oxygenatedmultiple methyl-branched acids such as17-hydroxy-2,4,6,8,10,12,14,16,-octamethyl C40 acid found insulfolipids.

Genes Involved in Mycolic Acid Biosynthesis

The biosynthesis of mycolic acids involves fatty acid chain elongation,desaturation, cyclopropanation of the olefin and a Claissen-typecondensation. The genes involved in cyclopropanation are cma1, cma2. Themethoxymycolate series found in M. tuberculosis contains a methoxy groupadjacent to the methyl branch, in addition to the cyclopropane in theproximal position. A series of four methyl transferase genes was cloned.The mm4 methylates the distal olefin. The multifunctional fatty acidsynthase (FAS) (type 1) catalyses not only the synthesis of C16 and C18fatty acids but also elongation to produces C24 and C25 fatty acids.Cloning and sequencing of the synthase gene revealed a 8389 bp ORF. Thedomain organization is much like a head to tail fusion of the two yeastFAS subunits; acyl transferase (AT)-enoyl reductase (ER)-dehydratase(DH)-malonyl/palmitoyl transferase-acyl carrier protein (ACP) fused with□-ketoreductase (KR)-□-ketoacyl synthase (KS).

The MAS gene encoding mycobacterial mycocerosic acid synthase is a dimerof the FAS gene. The cloning and sequencing of the MAS gene revealed thedomain organization in the following order: KS-AT-DH-ER- KR-ACP. Thepurified MAS shows a preference for elongation by four methylmalonyl CoAunits reflecting the natural composition of mycocerosic acids. FAS andMAS are also involved in the biosynthesis of phthiocerol andphenolphthiocerol which involve elongation of preformed n-C20 fatty acylchains or an acyl chain containing a phenol residue at the ω-end. Thecluster of five genes, ppsi1-5 encode the multifunctional enzymes(Fernandes N D et al., Gene 170: 95-99 (1996) Mathur M et al., J. Biol.Chem. 267:19388-19395 (1992) Yuan Y. et al., Proc. Natl Acad. Sci. U.S.A92: 6630-6634 (1995)).

Tumor cells are cotransfected with SAg-encoding DNA and nucleic acidsencoding the biosynthesis the above microbial products. The transfectedtumor cells acquire significant additional immunogenicity. These cellsare prepared as in Example 1-3. They are useful in vivo as apreventative or therapeutic antitumor vaccine (Examples 5 15, 16 18-23.They are also useful ex vivo to immunize T or NKT cells to produce apopulation of effector T or NKT cells for adoptive immunotherapy ofcancer (Examples 2-5. 15, 16. 18-23).

22. SAg-Ganglioside or SAg-Galactosylceramide Complexes Formed afterTransfection of Tumor Cells with DNA Encoding SAgs: Complete BacterialAntigen System Recognized by CD1 Receptors Capable of InducingAnti-Tumor Effects

SAg-encoding nucleic acid transfected into tumor cells express SAg onthe tumor cell surface which is bound to cell surface ganglio sideswhich are tumor associated antigens, oncogene product such as EGF orIGF. In this way the tumor associated antigen is capable of recognitionand interaction with host T cells and macrophages and of evoking apotent immune response. The SAg is also bound or associated with the CD1receptor alone or associated with the glycosphingolipid tumor associatedantigen.

SAgs have a natural affinity for glycosphingolipids on cell membranes.Enterotoxin-producing-bacteria secrete enterotoxins which in theirprecursor state are bound to cell membranes in dimeric form. Enterotoxintransfected tumor cells induce an anti-tumor response by expressing thetumor cell surface antigen in association with the SAg. Bound to thetumor cell membrane, the SAg may be in dimeric form associated with theceramide lipophilic anchor domain of a glycosphingolipid tumorassociated antigen. Likewise, the SAg may be associated with thecarbohydrate moiety or the ganglioside which protrudes from the cellsurface. It may also be secreted in monomeric or dimeric form fused tomembrane associated tumor antigen, oncogene product or receptor. If thetumor associated glycosylceramide, glycoprotein antigens, or glycolipidantigen with or without SAg are presented on CD1 receptors, then NKTcells may generate the predominant T cell response. However theclassical T cell system is also responsive.

These constructs are produced and used as a vaccine against establishedtumor by protocols given in Examples 2-5, 15, 16 18-23.

23. Nucleic Acids Encoding CD1 Receptors

Nucleic acid encoding the CD1 receptor is transfected into tumor cells,resulting in expression of the CD1 receptor on the tumor cell surface.Promoters of CD1 synthesis are also useful in this invention. The humangenome includes five CD1 genes (A-D)which also function in antigenpresentation to T cells (Calabi, F et al., CD1: From Structure toFunction in Immunogenetics of the Major Histocompatibility Complex,Srivastava, R et al., eds, VCH publishers, New York, N.Y., 1991). Inmice, two homologous proteins (mCD1.1 and 1.2) have been characterizedand map to chromosome 3. The human CD1 genes are located on chromosome1q221-q23 in the order D-A-C-E from the centromere on a 190 kb segmentof DNA. With the exception of CD1B, they are all in the sametranscriptional orientation. They are evenly spaced in the complex withone exception: CD1D and CD1A are spaced two to three times farther apartthan the average. The products of CD1A, -B and -C genes have beendefined serologically. The products of CD1D and CD1E are unknown. Theyshare a highly conserved exon domain which is homologous to the□2m-binding domain (a3) of MHC class I antigens. The CD1 molecules arenot polymorphic and apart from CD1D, are noncovalently associated with□2m in a TAP-independent manner. Complex alternative splicing of CD1genes results in tissue specific forms of the protein, which can beintracellular, membrane bound, or secreted. In cells infected withmycobacteria, the CD1 molecule binds and presents a mycobacterialmembrane component, mycolic acid. Surface CD1 molecules present longerpeptides than those normally found on class I molecules. Whether CD1molecules can also present peptide antigens is still unclear althoughthis has been shown for at least one member of the CD1 family.

Tumor cells are transfected with nucleic acid encoding the CD1 receptor.Nucleic acid encoding cell wall or cell membrane associatedglycosylceramides or α branched, □ hydroxy long-chain fatty acids foundin mycobacteria and other bacteria are cotransfected into the CD1transfected tumor cells. The tumor cell therefore displaysglycosylceramides bound to the CD1 receptor. Using site directedmutagenesis, DNA encoding the CD1 receptor is provided along with DNAencoding a SAg binding site. This SAg binding site consists of key aminoacids from the SAg receptor or from the SAg binding sites on (i) MHCclass II chains or (ii) the TCR V□ region. This may consist of aglycosphingolipid sequence (sensitive to endoglycoceramidase) present onsome mammalian cells. The glycosylceramide used to bind to the CD1receptor will have an exposed SAg binding site which is sensitive toendoglycoceramidase, an enzyme from Rhodococcus which specificallycleaves the glycosyl moiety from glycosphingolipids. Other ceramidasesbreak up sphingolipid into fatty acids and sphingosine.

These tumor cells transfectants are prepared as in Examples 1 and 2.They are used in vivo as a preventative or therapeutic antitumor vaccineas in Example 14-16, 18-23. They are also useful ex vivo to produce apopulation of tumor specific T or NKT cells for adoptive immunotherapyof cancer (Example 2-5, 7, 15, 16, 18-23).

24. DNA Encoding Streptococcal M Proteins and DNA Encoding Protein A orits Fc and VH3 IgG binding Domains Transfected into Tumor Cells Alone orSAg DNA

The streptococcal M proteins are type-specific and act as protective orvirulence factors. M protein genes are members of a larger emm-like genefamily, such that many S. pyogenes strains express more than one M-likeprotein. DNA encoding the streptococcal M protein and DNA of the largeremm-like family are transfected into tumor cells (Kehoe M. A.,“Cell-Wall Associated Proteins in Gram-Positive Bacteria,” In: BacterialCell Wall, Ghuysen J M et al., eds, Elsevier, Amsterdam, 1994).

In addition, DNA encoding protein A and its domains as well as DNA ofthe streptococcal fcrA 76 gene located upstream of the emm-like gene aretransfected into tumor cells individually or together to cause theexpression of IgG FcR- and VH3 IgG-binding domains (Kehoe MA, supra).DNA encoding SAg is cotransfected into the same tumor cells to produce atumor cell expressing any combination of M protein, protein A and a SAg.Such cells are used in vivo as preventive vaccines or as therapeuticvaccines against established tumors. See Examples 1-5, 11, 15,16, 18-23.They may also be used ex vivo to induce populations of active tumorspecific effector T cells that are then used in adoptive immunotherapySee Examples 2-5, 7, 15-16, 18-23. 25.

Nucleic Acids, Bacterial Cells and Phage Displays Mimicking SAgs

Because of circulating naturally occurring antibodies in humans, nativeor mutated SAgs that are administered parenterally are not likely toreach the appropriate receptors on T cells or tumor cells. To solve thisproblem, mimic oligonucleotides are prepared—these mimic SAgs in theircapacity to bind SAg receptors. Since no natural antibodies are directedto these compositions, they will not be prevented from reaching specificSAg receptors in vivo.

SAg receptors are used to screen oligonucleotides for their ability tomimic SAg binding. Useful receptors for such screening include thosedescribed herein (as expressed on tumor cells) and T cell TCR V chains.For example, pools of oligonucleotides are tested for their binding to,and affinity for, immobilized SAg receptors using nucleotidechromatography technology well known in the art. Once these highaffinity binding oligonucleotides are identified, they are isolated (or,following sequencmg, may be synthesized) and administered to a host.Also included here is a bifunctional oligonucleotide-peptide chimericmolecule that binds specifically to the SAg receptor on tumor cells aswell as the V□ region of the TCR. Such an oligonucleotide will bindsimultaneously to tumor cells and T cells (in the process of activation)to produce an anti-tumor response. An oligonucleotide-protein constructis prepared consisting of (a) a peptide sequence of enterotoxin A thatbinds to the TCR and (b) an oligonucleotide that binds to SAg receptoron tumor cells. The peptide portion of this construct should be devoidof MHC class II binding sites in order to minimize undesired binding ofthe molecule to class II structures upon administration in vivo.

In another embodiment, the nucleic acid portion of the chimeric moleculebinds to the TCR while the peptide consists of a non-enterotoxin ligandthat is specific for the SAg receptor on tumor cells. This construct hasthe advantage of lacking any binding site for natural antibodies.

Yet another additional chimeric molecule consists of an oligonucleotideportion specific for the class II (or (chain and a secondoligonucleotide or a peptide specific for the TCR V□ chain.

Methods for preparing these constructs are given in Examples 5, 13.These constructs are especially useful for targeting tumors in vivowhile also promoting a T cell anti-tumor response. See Examples 18-23.However, these chimeric molecules may also be used ex vivo in theproduction of tumor specific effector T cells capable of inducing, oreffecting, an anti-tumor response when administered to a tumor bearinghost. See protocols in Examples 2-5, 15, 16 18-23.

SAg and GlycosylCeramide Co-Expression

This may be accomplished using intact bacteria or phage displayapproaches. Since the precursors and substrates of theglycosyltransferases are not readily available in most mammalian cells,it is more convenient to induce dual expression of GalCer and SAgs inbacteria, for example Sphingomonas paucimobilis, which naturally produceGalCer. Hence, nucleic acid encoding a SAg is transfected into thisbacterium together with a suitable promoter well known in the art. Thebacterium produces both GalCer and SAg. By ensuring that the SAgcontains one or more glycosylation sites (by using the appropriatenucleic acid sequence), a glycosylated SAg is produced. Such a SAg bindsto the glycosyl ceramide, e.g., GalCer to form a conjugate that isexpressed on the bacterial surface of is secreted. In either form, sucha SAg-GalCer conjugate can sensitize NKT cells to produce an anti-tumorresponse. In addition, phage or plasmids encoding the appropriatetransferase are transfected into low virulence Staphylococcus specieswhich also produce enterotoxins. The bacterium acquires the capabilityof expressing GalCer on its surface. These bacterial constructs andcompositions are used in vivo in a tumor bearing host to produce ananti-tumor response in protocols given in Examples 5, 13, 15, 16 18-23and Detailed Description Section 19. They are also are used ex vivo toactivate NKT cells or T cells to differentiate to tumor specificeffector cells for use in adoptive immunotherapy of cancer by protocolsin Example 1, 2, 14-16, 18-23).

Phage display technology is used to target selected SAg sequences totargets in vivo. The selected peptide is used as a binding sequences inlieu of the full-length polypeptide. This permits elimination from theconstruct of the antigenic portion of the SAg to which naturalantibodies are directed. Cloned genes are expressed as part of phagecoat proteins, for example, as fusions with the gene III protein (gIIIp)or the gene vIII protein (gVIIIp). In addition to the displayed geneproduct, the phage genome (of each particle) includes the gene encodingthis product.

Phage display is preferably done using the filamentous phage f88-4 andcomprises forming a fusion that results in the C terminus of the“selected” (i.e., inserted gene's) product and the N terminus of thephage protein gVIIp. Peptides of various enterotoxins are expressed inthe phage display—most preferably peptides that bind to the SAg receptoron colon carcinoma cells. These peptides retain their capacity to bindto the TCR and to activate T cells. Also contemplated within thisinvention is phage display of SAg plus nucleic acid encoding synthesisof GalCer and/or the Gal epitope. DNA for synthesis of GalCer ispreferably isolated from Sphingomonas paucimobilis; DNA encoding thegalactosyl transferase for synthesis of Gal is preferably isolated fromKlebsiella aerobacter, Serratia, E. coli and Salmonella organisms whichnaturally produce and express these epitopes. The phage displays areadninistered in vivo and are capable of initiating a potent immuneresponse to the tumor using the protocols described in Examples 5 and 13and Section 19, above. These preparations are also useful for activatingT cells or NKT cells ex vivo to produce a tumor specific effector cellsfor use in adoptive immunotherapy (Examples 2-5, 14-16, 18-23).

Viral infection of a host cell having the galactosyl transferase resultsin the shedding of virions that express the Gal epitope. When a hostmammalian cell has been transfected with nucleic acid encoding SAg, thevirus can coexpress the Gal epitope and the SAg on its surface. Such aviral construct is administered in vivo to achieve a therapeutic effect,or, in another embodiment, is employed ex vivo to produce tumor specificeffector T or NKT cells for use in adoptive immunotherapy of cancer(Examples 2, 3, 7, 15, 16, 18-23).

26. Combining SAgs with Enterotoxin Precursors (Cell-Bound Dimers andOligomers) and with Enterotoxin Promoters and Transcriptional RegulatoryGenes

Cell-Bound SAg Dimers and Oligomers

Staphylococcal enterotoxins are present in the membrane of enterotoxinproducing bacteria in dimeric form and retain potent enterotoxin-likeactivity when isolated from the membrane. It is in this membrane-boundform that enterotoxins are combined with tumor associated antigens oroncogene products and presented to the T cell system. The dimerizationof the enterotoxins may promote clustering for more effectivepresentation to T cells. Indeed, dimerization or polymerization ofenterotoxins or the introduction of tandem repeats of the SAg bindingsites for TCR and MHC class II may be achieved by (1) site directedmutagenesis of the enterotoxin plasmid and (2) introduction of sequencesfor gene amplification, tandem repetition and/or recombination or by (3)introduction of enzymes for peptide chain elongation. The duplicationmay be at the level of the bacterial operon including itstranscriptional regulators, using methods well described in the art.Modified plasmid is DNA is introduced into the target tumor cells orinto accessory cells, either or both of which are useful in vivo as apreventative or therapeutic vaccine (Examples 1, 2, 15, 16, 18-23). Suchgenetically transformed cells may also be used ex vivo to produceeffector T or NKT cells for adoptive immunotherapy (Examples 1, 2, 7,15, 16, 18-23).

SAg agr Locus (Accessory Gene Regulator) and Other Bacterial Genes andElements

At least 15 gene coding for potential virulence factors in S. aureus areregulated by a putative multicomponent signal transduction systemencoded by the agr/hld locus. The synthesis of at least 14 exotoxins andenzymes in S. aureus is regulated by a set of trans-acting elements fromagr. The agr gene coordinately controls the expression of exfoliatintoxin, toxic shock syndrome toxin, a, b, d toxins, enterotoxin B,lipases and nucleases (Balaban, N. et al., Proc. Natl. Acad. Sci. U.S.A92:1619-1623 (1995)). These proteins are members of the histidineprotein kinase family of regulators and control a number of virulencedeterminants (Balaban supra, Novick R P, Meth Enzymol. 204: 587-637(1991)). Compared to wild-type, agr and hld mutants have decreasedsynthesis of extracellular toxins and enzymes (such as α-, □-, andγ-hemolysins, leucocidin lipase, hyaluronate lyase and proteases) whilehaving increased synthesis of coagulase and protein A. The agr geneconsists of two divergent transcriptional units driven by promotersnamed P2 and P3. The P2 transcript includes four open reading framesreferred to as agrA, B, C, and D, all four of which are required to forthe agr response. The peptides predicted for agrA and agrc resemble theresponse regulators and signal transducers of the two-componentbacterial signal transduction systems. The primary function of thee fourgenes discussed above is to activate two promoters; the P3 transcript,RNAIII, however is the actual effector of the exotoxin response. RNAIIIactivates transcription of secretory protein genes and repressestranscriptions of surface protein genes. As a global regulatory system,agr, controls the post-exponential production of exoproteins such astoxins, hemolysins, and exoenzymes. agr is a complex polycistronic locusthat encodes a two-component signal transduction pathway that activatestranscription of a regulatory RNA molecule that in turn activatestranscription of the exoprotein genes.

Thus, transcriptional regulation of the enterotoxin B gene as well asSED, SEC and staphylococcal capsular polysaccharide gene involves theagr product. (agr does not regulate SEA expression).

The promoter region of SEA is localized by primer extension analysis.The 5′-end of SEA mRNA is localized 86 bp upstream of the translationalinitiation codon. A DNA region with good agreement with canonicalpromoter sequences was observed beginning 8 base pairs upstream of theapparent transcriptional start site. No DNA upstream of the 35 bp regionis required for transcription. Both the agr gene and the SEA promoterhave been cloned (Peng, H. L. et al., J Bacteriol. 170:4365-4372 (1988);Borst, D. W. et al., Infec. Immun. 61:5421-5425 (1993)). The xpr locusand the agr locus interact at the genotypic level; agr is autoinduced bya proteinaceous factor produced and secreted by the bacteria and isinhibited by a peptide from an exotoxin-deficient S. aureus mutantstrain. The inhibitor, RIP, competes with the activator, RAP. When givenas a vaccine, RIP may prove useful as a direct inhibitor of virulence.

A chromosomal locus (sar) distinct from agr, encodes a DNA-bindingprotein that is important in regulation, and is required for expressionof S. aureus exoproteins including enterotoxin, toxic shock syndrometoxin, hemolysin and staphylokinase. Transcription of Protein A issuppressed by sar and agr. A list of plasmids containing bacterialvirulence factors useful in this invention is disclosed in Table 49, p.223 of Patrick, S. et al., Immunological and Molecular Aspects ofBacterial Virulence, John Wiley and Son, New York, N.Y. 1995. Thisinvention contemplates the use of the Staphylococcal enterotoxinpromoters and transcription factors that activate the enterotoxinbiosynthetic cycle. Several Staphylococcal promoters have beenidentified (Novick, supra). This invention also contemplates the use ofthe peptide activator RAP which induces agr as well as the peptideinhibitor RIP which induces or represses RNA III.

SAg-encoding nucleic acid is fused in-frame with Staphylococcus agrnucleic acid and introduced into tumor cells or accessory cells (or thetwo are cotransfected into these cells). In another embodiment,SAg-encoding nucleic acids placed under the control of an enterotoxinpromoter, and this construct is introduced into tumor cells or accessorycells. The agr gene is especially useful because it can be linked to aninducible promoter such as that for corticosteroids or themetallothionein promoter, allowing it to be activated in a controlledmanner by exogenous administration of the inducing to the host.

Methods for introducing the above genes into tumor cells are describedin Example 1, 2, 11. The use of such cells in vivo as preventative ortherapeutic vaccines are discussed in Examples 15, 16, 18-23. Use ofthese genetically transformed tumor cells ex vivo to induce effector Tor NKT cells for adoptive immunotherapy is described in Examples 2, 3,7, 15, 16, 18-23.

27. Combining SAg with Oncogenes, Protooncogenes, Amplified Oncogenes,Transcription Factors or Tumor Markers

In one embodiment, the nucleic acid encoding a SAg is fused in-frame tooncogene or protooncogene nucleic acid in tumor cells or accessory cellsto produce a chimeric nucleic acid which is expressed in, or on thesurface of, the cell. This fused gene may be rendered inducible byjudicious choice of a promoter or other regulatory sequence. Preferably,such an inducible promoter is induced by a hormone or a metal. Aregulatory element, such as one activated by interferon or a cytokine(e.g., Jak or a STAT), may be included in this construct. In anotherembodiment, the nucleic acid encoding SAg is fused in frame to nucleicacid encoding an oncogene which can be amplified markedly. The fusedconstruct is introduced into tumor cells or accessory cells. Anamplified “unit” is initially much larger than the size of the actualgene of importance to the oncogenic event(s) (Hellems, R E, GeneAmplification in Mammalian Cells, Marcel Dekker, New York, N.Y. ). Thusa silent gene is co-amplified with one or more genes expressed on anamplicon. This is a preferred site for the inserting gene clusterswherein one gene encodes a SAg, others encode the enzymes of LPS lipid Abiosynthesis, optionally together with their native promoters oroperons.

Transcription Factors and Amplified Oncogenes

Oncogenes are frequently amplified in human tumors and cultured cancercells. This is more characteristic of solid tumors and relatively rarein lymphoid malignancies. DNA amplification was first observedcytogenetically a double minute chromosomes (DMs) or homogeneouslystaining regions (HSRs) but today, direct DNA analysis (Southernblotting) or molecular cytogenetic methodologies, such as fluorescencein situ hybridization (FISH) and comparative genomic hybridization (CGH)can be applied. DMs are episomal forms of amplified DNA that generallylack centromeres and are unequally distributed between daughter cells atmitosis. They appear as isodiametric extrachromosomal bodies stainablewith all chromatin dyes. HSRs are chromosomally integrated forms ofamplified DNA. They represent either the replacement of the normalchromosome banding pattern with an extended region of homogenousstaining or the insertion of such a region into an otherwise normallybanded chromosome. DMs and HSRs tend to be mutually exclusive and arepotentially interchangeable manifestations of the amplified DNA. Thus,DMs can potentially integrate into distant chromosomal sites to generateheritable HSR. Of 22 human tumors analyzed, 91% contained DMs only, 6.5%contained HSRs and 2.5% contained both. In solid tumors of epithelialorigin, DMs and HSR were found in 40% of breast carcinomas, 17% of nonsmall cell carcinoma of the lung, 18% of stomach and esophageal cancersand 15% of uterine carcinomas.

The overwhelming majority of oncogene amplifications in human tumorsaffect the Myc oncogene family. In small cell lung cancers all threemembers of the Myc family, c-myc, N-myc and L-myc can be involved. Mycamplification is associated with a more invasive and more metastaticphenotype. N-myc amplification is seen in neuroblastoma and isassociated with the late stages and poor prognosis. The amplificationunits on chromosome 11q13 are seen in (a) breast cancer, (b) squamouscell carcinoma of the head and neck, lung, and esophagus and (c) bladdertumors. The amplification extends for over 1.5 megabase pairs of DNA andincludes two bona fide oncogenes: FGF3 and FGF4. It also includes theBcl-1 CCND1 (cyclin D1) as well as the EMS1 gene that encodes the humanhomologue of cortactin. CCND1 has a critical role in amplified DNA sinceits expression is increased as a consequence of amplification. The othermajor targets for amplification are the genes encoding the EGF receptor(ErbB1/Her1) and the related ErbB2/Her2. Both genes are amplified inbreast cancer and other malignancies. ErbB2 is associated with estrogenreceptor-negative breast cancers and poor prognosis.

Members of the myc gene family are activated in several human tumors asa result of DNA rearrangements through chromosomal translocations orgene amplification. When overexpressed, all myc genes complement mutantc-ras oncogenes in the transformation of primary rat embryonic cells andtransform Rat 1-A cells without assistance of other oncogenes.Stimulation of cellular myc expression levels or changes inpost-translational modification of myc proteins have been followingexposure of cells to many growth promoting stimuli. These featuressuggest that the myc proteins participate in the final steps ofmitogenic signal transduction. The myc proteins act as transcriptionfactors involved in activation and/or repression of target genes. Inneuroblastoma, a group whose tumors are generally near diploid ortetraploid with chromosome 1p deletion(LOH) and N-myc amplification havea generally poor response to treatment and a poor prognosis. Genomicamplification of the N-myc cellular oncogene is present in approximately40% of cases of childhood neuroblastoma and correlates withhistopathological signs of advanced disease. This genomic N-mycamplification appears to be associated with tumor progression ratherthan tumor initiation since early stage tumors rarely exhibit M-mycgenomic amplification. Similarly the c-myc family of protooncogenesincluding N-myc and L-myc are amplified in small cell carcinoma of thelung.

The amplified oncogenes useful in the present invention include genesencoding transcription factors. The preferred nucleic acids for use inthe present invention are c-myc, N-myc, c-abl, c-myb, c-erb, c-Ki-ras,N-ras. N-myc (amplified 5-1000 fold in neuroblastoma) is preferred.SAg-encoding nucleic acid is cotransfected into tumor cells or accessorycells with amplified oncogenes. The N-myc and L-myc genes have beencloned as c-myc homologous amplified oncogenes from human tumors. In oneembodiment, SAg-encoding nucleic acid is fused in frame with nucleicacid encoding oncogenic transcription factors such as FOS, JUN. MYC, MYBand ETS. In another embodiment, such nucleic acid is cotransfected withSAg-encoding nucleic acids. Either of such constructs is introduced intotumor cells or accessory cells. Proteins that interact with FOS and JUNare given in Table 1 p. 157 of Peters G et al., Oncogenes and TumorSuppressors, Oxford University Press, Oxford UK 1997, incorporated byreference.

bcr/abl Gene

SAg-encoding nucleic acid is fused in frame or cotransfected withnucleic acids encoding the following agents and transfected into tumorcells and fused to oncogenic nucleic acids encoding chimeric proteinscapable of immunizing the tumor bearing host. An ideal candidates forsuch fusions is the bcr-abl gene which express the bcr/abl protein inchronic myelogenous leukemia (CML). The c-abl oncogene is amplified inchronic myelogenous leukemia. Scherle P A et al., Proc. Natl. Acad. Sci.U.S.A 87: 1908-1917 (1990) Heisterkamp N et al., Nature 344: 251-253(1990). Abnormalities in the structure and expression of the human c-ablcellular oncogene have been associated with Philadelphiachromosome-positive CML which is present in more than 90% of cases. Thisaberrant chromosome marker is generated by a reciprocal translocationbetween chromosomes 9 and 22 in which the c-abl oncogene is translocatedfrom the distal end of the q arm of chromosome 9 to a relativelyrestricted 5-6kb region on chromosome 22 termed the breakpoint clusterregion (bcr). This translocation creates a fusion gene that istranscribed as an 8 kb bcr-abl RNA that encodes the aberrant bcr-ablfusion protein product (P210) observed in CML cells. The bcr-abl fusionproduct has enhanced tyrosine kinase activity compared with the normalp145 c-abl product. Abnormalities in the structure and expression of thec-abl cellular oncogene have not been described in any type of humanmalignancy other than CML and Ph positive acute lymphatic leukemia. Geneamplification correlates with progression of malignancy.

EGF Receptor Genes

SAg-encoding nucleic acid is fused in frame to the nucleic acidsencoding the EGF receptor (EGFR) (Ulrich A et al., Nature 309: 418-421(1984)). The EGFR is the prototype of four-member receptor family. EGFRis frequently overexpressed or mutated in several different types ofhuman tumor. For instance, the EGFR is amplified in 20-40% of humanglioblastomas and a variety of epithelial tumors including head and necksquamous cell carcinomas, breast tumors, esophageal tumors andurogenital tumors. Amplification was accompanied by overexpression ofthe EGFR.

The erbB2 (her2/neu) Oncogene

SAg-encoding DNA is fused in frame to DNA encoding a tumor marker suchas PSA, c-erbB2(neu), her2/neu, bcl-2 and Brca-1. The principalamplified and functional genes in breast cancer are the growth factorreceptor-erbB2, the nuclear transcription factor c-myc, and the genesencoding cell cycle kinase regulatory genes termed cyclin D1 and cyclinEG. Gene amplification is thought to proceed via the initial formationof extrachromosomal, self replicating units (double minute chromosomes)that become permanently incorporated into chromosomal regions where theyare called homogeneously staining regions (HSRs) as described above.

The human counterpart of the oncogene neu known as her2 encodes aprotein of the same family as the EGFR. This family of genes has beencloned. Its products belong to a family of receptor tyrosine kinaseseach with a transmembrane domain, a cysteine-rich extracellular domainand an intracellular catalytic domain. They act as receptors for severalpeptide growth factors such as EGF, TGF(and neuregulins. The activatedreceptors are then able to bind to proteins containing src-homology-2(SH₂) domains. The SH₂ domain proteins recognize and bind to specificphosphotyrosine-containing sequences of the activated receptor. TheseSH₂ containing adapter molecules then trigger downstream signallingpathways, ultimately resulting in gene activation.

The erbB2 (neu/Her2) gene maps to chromosome 17p21 and codes for a 185kDa transmembrane glycoprotein related to, but not identical to the EGFreceptor (Schechter A L et al., Science 229: 976-978 (1985), Bargmann CL Nature 319: 226-230 (1986), Hung M C et al., Proc. Natl. Acad. Sci.U.S.A 83: 261-264 (1986), Yamamoto T et al., Nature 319: 230-234(1986)). The EGFR bears sequence homology with the erbBl product. TheerbB2 gene is activated by a point mutation which mutates amino acidresidue 664 from valine to glutamic acid; this change is associated withtransforming its ability. The genes are called erbB, (erbB1, EGFr),erbB2, (neu/Her-2). erbB3(HER-3) and erb B4 (HER-4). Amplification andoverexpression of erbB2 has been found in a variety of human tumorsincluding carcinomas of the breast, ovaries, colon, lung, liver,stomach, kidney, esophagus, salivary gland, and bladder. Genomicamplification of the neu (C-erb-2) or HER2 cellular oncogene and proteinoverexpression has been documented in approximately 30% of primary humanbreast cancers and may correlate with advanced disease and a relativelypoor prognosis. More than 50% of all ductal carcinomas in situ of thelarge cell type express HER2. Amplification occurs in approximately 20%of invasive breast carcinomas. Thus, it is thought that HER2amplification increases the growth rate but not the metastatic potentialof tumor cells.

A third member of the EGFR family is ERBB3which is present in some humanbreast cancers with high expression correlating with lymph nodemetastases. Overexpression of ERBB3 has been observed in epidermoidcarcinoma of the larynx and esophageal carcinoma. ERBB4, a fourth memberof the EGFR family, was overexpressed in a human mammary tumor cellline. Fisk et al. (J. Exp. Med. 181: 2109-2117 (1995)) described animmunodominant epitope of HER/neu that is recognized by ovariantumor-specific cytotoxic T lymphocytes. This epitope is useful in thisinvention. Failure of coexpression of a heterodimeric partner orcoinduction of a suppressor phosphatase would explain the lack ofimmunogenicity of c-erbB2 in mice in nude mice.

Additional oncogenes, protooncogenes and tumor markers which would becandidates for the fusion in accordance with this invention are w PSA,c-erb B2(neu), Her2/neu, bcl-2, Brca-1. Viral and non-viral oncogenesand protooncogenes which are overexpressed in tumor cells are shown inTable 9.2 and 9.1, p. 171-172 of Franks et al., supra). The functionsofthe various oncogenes is shown in Table 9.6, p. 186, of Franks et al.

IGF Receptor Genes

SAg-encoding nucleic acid is fused in frame with nucleic acids encodinginsulin-like growth factor (IGF) receptors (IGFRs)and transfected intotumor cells. The IGFR gene is a tyrosine kinase containing transmembraneprotein that plays an important role in cell growth control. There is asingle IGF1 receptor gene with a complete coding sequence contained in21 exons (Abbott A M et al., J. Biol. Chem. 267: 10759-10763 (1992);Scott J et al., Nature 317: 260-262 (1985); Liu J et al., Cell 75: 59-63(1993)).

IGF1R is expressed at high levels in breast cancer, and amplification ofthe IGF1R gene has been observed. IGFs play a significant auxiliary rolein tumor growth by suppression of apoptosis. The apoptotic effectoverexpressed myc is overcome by IGFs. Thus, IGFs facilitate tumorgrowth by suppression of apoptosis.

Fibroblast Growth Factor (FGF) Receptor Genes

SAg-encoding nucleic acid is fused in frame to nucleic acids encodingfibroblast growth factors receptors (FGFs) and transfected into tumorcells. FGF receptor are also be important for the vascularization ofcertain types of tumors. The expression of FGF1 has been shown to beassociated with a switch to an angiogenic phenotype during thedevelopment of a fibrosarcoma. Overexpression of FGF receptor by certaintumors may also contribute to their growth. FGF receptors have beenshown to be amplified in some breast cancers.

Platelet Derived Growth Factor (PDGF) Receptor Genes

SAg-encoding nucleic acid is fused in frame to nucleic acids encodingadditional tumor growth factors which are produced or overexpressed andtransfected into tumor cells or accessory cells. Growth factors includethose in the tyrosine kinase receptor families such as Platelet DerivedGrowth Factor A and B family (PDGF). PDGF A and B receptors areamplified in malignant glioblastomas in the malignant cells themselvesor the stromal cells (Fleming T P et al., Cancer Res. 52: 4550-4556(1992);Kumabe T et al., Oncogene 7: 627-632 (1992)). The nerve growthfactor (NGF), stem cell factor receptor (kit), colony stimulatingfactor-1 receptor (fms), neurotropin receptor family, transforminggrowth factor □ family, the WNT family, angiogenic receptors

Other Amplified Oncogenes

SAg-encoding nucleic acid is also fused to nucleic acid encoding thetyrosine protein kinases which are both membrane associated andtransmembrane as described in Table 9.4, p. 179 of Franks et al., supra.Additional chromosomal regions which are amplified in greater than 40%of cases included the 8q24 locus of the c-myc(O) gene, the 11q13 locusof the cyclin D (O), int2 (O), EMS-1 (O), BCL-1 (O), FGF-4 (O) GST (M),MEN1(S) genes, the 17q21 locus of the RARa (S), RARg (S), ERBAa (S),BRCA1 (S), NM23 (S), estradiol 17B dehydrogenase (S) ERG2 (O), HOX2,NGFR (O), WNT3 (O) and the 20q13 locus.

Nucleic acid encoding SAg is fused or cotransfected into tumor cellswith nucleic acid encoding the above oncogenes, amplified oncogenes andprotooncogenes, transcription factors and growth factor receptors. Thesetransfectants are prepared as in Examples 1. They are useful in vivo asa preventative or therapeutic vaccine (Examples 15, 16, 18-23). They arealso useful ex vivo for inducing tumor specific effector cells foradoptive immunotherapy (Examples 2-5, 7, 15, 16 18-23).

28. Combining SAg with Angiogenic Receptors and Growth Factor Receptors

SAg-encoding nucleic acid is cotransfected or fused in frame to nucleicacid encoding an angiogenic receptor such as VEGF and transfected intotumor cells. SAg nucleic acid is also fused to or cotransfected withnucleic acid encoding other angiogenic receptors such as v integrin,other integrins, cadherins or selectins and introduced into tumor cellsor accessory cells. SAg-encoding nucleic acid is also cotransfected intotumor cells or accessory cells with nucleic acids encoding angiogenicproteins such as VEGF. VEGF is produced by tumor cells and stroma, andits expression correlates with the degree of vascularization and gradeof malignancy. VEGF receptors, termed KDR and flt, are expressed mainlyby the tumor endothelium. Higher levels of VEGF are found in metastaticthan in non-metastatic colon cancers (Tischer E et al., J. Biol. Chem.266: 11947-11954 (1991). VEGF is especially useful here because it isoverexpressed in tumor cells at an early stage of tumorigenesis. Thepromoter of the VEGF gene lacks a TATA box, but has six GC boxes fortranscription factor SP-1 binding and also a site for AP-1 and AP-2binding. The expression of the gene is modulated by several growthfactors such as EGF. In some cell types VEGF expression is regulated byIL-1, FGF, PDGF. A common element, mediation of protein kinase C in theregulation of VEGF, has been suggested. VEGF is expressed as a disulfidelinked dimer. Long and short forms are generated by alternative splicingand are matrix bound or released, respectively. As a result of itsspecific effects on endothelial cell migration and proliferation, VEGFis a very potent and specific promoter of angiogenesis. Two wellcharacterized families of angiogenic factors act by binding to tyrosinekinase receptors that have two or three immunoglobulin-like domains, andVEGF binds to two related receptors with seven immunoglobulin-likeextracellular domains.

The TRKA oncogene codes for a receptor for nerve growth factor (NGF).The TRKA gene has been found fused to genes that code for proteins thatform dimers in cells leading to the synthesis of a constitutivelydimerized and active tyrosine kinase. TRKA may have a tumor suppressorfunction since its expression in neuroblastoma correlated inversely withn-myc gene amplification. Coexpression of mRNA for TRKA and the lowaffinity NGF receptor in neuroblastoma correlated with a favorableprognosis.

Nucleic acid encoding SAg is fused to nucleic acid encoding the aboveangiogenic factors or receptors and introduced into tumor cells;alternatively, the two nucleic acids are used to cotransfected tumorcells. These transfectants are prepared as in Example 1. They are usefulin vivo as a preventative or therapeutic antitumor vaccine (Examples 15,16, 18-23). They are also useful ex vivo for inducing tumor specificeffector cells for adoptive immunotherapy of cancer (Examples 2-5, 7,15.16 18-23).

29. Combination of SAg with Cell Cycle Protein

SAg-encoding nucleic acid is fused in frame to nucleic acid encoding acell cycle protein such as a cyclin which is overexpressed in tumorcells. Examples of these cell cycle proteins which are preferred forsuch fusions are Cyclins A, B, D1, E. These proteins are generallycomplexed to kinases or transcription factors at critical checkpoints inthe cell cycle. The cyclins, CDKs and their inhibitors are shown inTable 1. p193 of Peters G et al., supra.

In another embodiment, nucleic acid encoding SAg is cotransfected intotumor cells with nucleic acid encoding a cell cycle protein as above.These transfectants are prepared as in Examples 1. They are useful invivo as a preventative or therapeutic antitumor vaccines (Examples 15,16, 18-23). They are also useful ex vivo for inducing tumor specificeffector cells for adoptive immunotherapy of cancer (Examples 2-5, 7,15.16 18-23).

30. Combining SAg with Tumor Suppressor Genes, p53 or DevelopmentalGenes

SAg-encoding nucleic acid is fused in frame with tumor suppressor geneDNA and the fused nucleic acid is introduced into tumor cells oraccessory cells. Alternatively, the two nucleic acids are used tocotransfected these cells. Examples of such tumor suppressor genes areshown in Table 9.7 p.187 of Franks L M et al., supra. Examples ofmutated tumor suppressor genes include the APC and MCC genes and theirisoforms, the DCC gene in colon cancer, the BRCA1 tumor suppressor genein breast cancer and the DPC gene in pancreatic cancer.

The p53 gene and its mutations are also useful in this embodiment. Alist of p53 responsive elements and associated proteins useful in thisinvention is given in Tables 1 and 2 pp. 267-269 of Peters G et al.,supra.

In another embodiment, nucleic acid of developmental genes is used inplace of tumor suppressor or p53 genes. Examples of such developmentalor differentiation genes are wnt and fwt genes. Transfectants areprepared as in Examples 1. They are useful in vivo as a preventative ortherapeutic antitumor vaccine according to Examples 15, 16, 18-23). Theyare also useful ex vivo for inducing tumor specific effector cells foradoptive immunotherapy of cancer (Examples 2-5, 7, 15, 16, 18-23).

31 . Combining SAg with Cell Surface Glycoproteins or their Receptors

SAg-encoding nucleic acid is fused in frame with a nucleic acid encodinga cell surface glycoprotein and or its receptor and the fused nucleicacid is introduced into tumor cells or accessory cells. Alternatively,the two nucleic acids are used to cotransfected these cells. Examples ofthese glycoproteins or receptors include integrins, vitronectinreceptors, laminin receptors, cadherins, tenascin and CD44 and isoforms,VCAM-1, P-Selectins, E-Selectin, NCAM and MCAM. Transfectants areprepared as in Example 1. They are useful in vivo as a preventative ortherapeutic antitumor vaccine according to Examples 15, 16, 18-23). Theyare also useful ex vivo for inducing tumor specific effector cells foradoptive immunotherapy of cancer (Examples 2-5, 7, 15, 16 18-23).

32. Combining SAg with Cytokines and Chemokines

SAg-encoding nucleic acid is fused in frame with nucleic acid encoding acytokines and chemokines, and the fused nucleic acid is introduced intotumor cells or accessory cells. Alternatively, the two nucleic acids areused to cotransfected these cells. Examples of chemokines and cytokinesthat are useful herein include RANTES, IL-5, IL-7, IL-12, IL-13, IFN(,TNF(and TNF(. Chemokines are small (typically 6-10 kDa) peptides thathave been divided into two classes designated C-C and CXC based on thesequence of the first two cysteine residues. The two families exhibitpreferences for different target cell types: C-C chemokines actprimarily on macrophages.

Chemokine gene expression is induced by the action of other growthfactors and cytokines and are actively expressed in solid tumors showinginflammatory involvement and macrophage or neutrophil invasion.Chemokines of the C-X-C class containing the amino acid sequence motifELR have demonstrable angiogenic activity which can be inhibited byC-X-C chemokines lacking the ELR motif. Therefore chemokine expressionby either tumor cells themselves or elicited from stromal cells by theaction tumor-derived growth factors, have the potential to regulatetumor growth by modulation of angiogenesis. G-CSF is a growth factor forgranulocyte precursors, and IL-2 is a growth factor for T cells. Nucleicacids encoding SAgs are fused or cotransfected into tumor cells withnucleic acids encoding the above cytokines, chemokines andchemoattractants. The transfectants are prepared as in Example 1. Theyare useful in vivo as a preventative or therapeutic antitumor vaccineaccording to Examples 15, 16, 18-23). They are also useful ex vivo forinducing tumor specific effector cells for adoptive immunotherapy ofcancer (Examples 2-5, 7, 15, 16 18-23

33. Combining SAg with Transcription Factors AP-1 and NFκA

Transcription factor genes may act as oncogenes. The jun family oftranscription factors bind specifically to AP-1 sites which confer theeffects of potent tumor promoting phorbol esters on responsive genes andspecifically bind to c-jun homodimers or c-jun/c-fos heterodimers. v-relencodes members of the NF-κB family of transcription factors.Transforming oncogenes such as v-ets and v-myb also encode transcriptionfactors.

The T cell signaling system responding to SAgs activates the JAK, TNF(TRAF), IL-2 and IL-12 pathway probably via NFκA activation. LPS has a Tcell stimulating effect and may fuse with SAg to produce additionalstimulation or epitope expansion. The NFA nucleic acids are fused to apromoter which activates sequences encoding the SAg receptor or thesequences encoding the key V□ domains binding SAgs or regions in the V□receptor which are activated by the SAgs.

SAg-encoding nucleic acid is fused in frame with nucleic acids encodinga transcription factor such as those above. Transfectants are preparedas in Example 1. These transfectants are prepared as in Example 1. Theyare used in vivo as a preventative or therapeutic antitumor vaccineaccording to Examples 15, 16, 18-23). They are also used ex vivo forinducing tumor specific effector cells for adoptive immunotherapy ofcancer (Examples 2-5, 7, 15, 16 18-23).)

34. SAgs Augment the Immunostimulatory Effects of Tumor AssociatedPeptides, Binary and Ternary Complexes

Bacterial SAg are presented to T cells via the MHC class II molecule bymultiple low affinity attachments, resulting in stimulation of the Tcell with very low concentrations of antigen. SAgs augment thepresentation of antigenic peptides to T cells without stericallyinterfering with each other's ability to bind and activate the TCR.These augmenting peptides are incorporated into the SAg structure. SAgsmay also bind to binary or ternary complexes of tumor peptide-MHC classI or tumor peptide-MHC class II complexes, either in solution or affixedto a TCR or the surface of an APC. In one embodiment, the SAg is firstbound to APCs or T cells followed by addition of complexes between MHCclass I or class II and tumor peptide. Alternatively, the SAg may firstbind to either cell-bound, soluble or immobilized MHC class I or classII molecules, after which the tumor peptide is added. This trimolecularcomplex is then presented to the T cell via the TCR. In anotherembodiment, SAg is first bound to an APC or to a TCR V□ chain on an NKTcell. Following this, CD1-glycosylceramide complexes are added andallowed to bind to NKT cell TCR V□ chain. SAg may be bound to first toCD1-glycosylceramide complexes in soluble form, affixed to CD1+ cells orNKT cells via the TCR. SAgs may be bound to CD1 complexes withglycosylceramide or a glycosphingolipid (with a conserved SAg bindingsite) in solution or when fixed to CD1+ cells or NKT cells.Alternatively, SAgs are bound to ternary complexes consisting ofCD1-glycosylceramide affixed to the NKT cell TCR or bound toCD1-glycosylceramide on APCs, in solution or immobilized, before it hasaffixed to the NKT TCR. SAg is alternatively bound to binary complexesof (a) CD1-glycosylceramide, (b) CD1-glycosphingolipid, (c) CD14-LPS or(d) MHC-tumor peptide complexes that have either a SAg receptor sequenceor a TCR V□ SAg-binding sequence.

The complexes described above are used in vivo as preventative ortherapeutic antitumor vaccines according to Examples 4, 15, 16, 18-23.They are also used ex vivo for inducing tumor specific effector cellsthat are then taken for adoptive immunotherapy of cancer. (See Examples2-5, 7, 14, 15, 16 18-23).

35. SAgs Combined with Products of Antigen Processing Pathways

A chimeric gene is prepared consisting of SAg-encoding nucleic acidfused in frame to nucleic acids encoding (a) the endoplasmic reticulum(ER) translocation signal peptide, (b) transmembrane domain, and (c)lysosomal targeting domain of LAMP-1. LAMP-1 is a type 1 transmembraneprotein localized predominantly to lysosomes and late endosomes. Thecytoplasmic domain of LAMP-1 contains the Tyr-Gln-Thr-Ile sequence thatmediates the targeting of LAMP-1 into the endosomal and lysosomalcompartments. The specific targeting of the SAg to the endosomal andlysosomal compartments allows SAg peptides to complex with MHC class IImolecules and enhance presentation.

The MHC class I presentation pathway operates on a three level system.At one level there is protein machinery dedicated to peptidemanufacture—the proteosome complex. The selective peptide transportersdeliver antigens into the ER. The class I molecules themselves exhibitvariable affinities for peptides. Genes clustered in the region of theclass II gene encode proteosome and transporter. SAg peptides aretransported into the ER—primarily through a transmembrane “tube”consisting of two polypeptide chains called TAP-1 (SEQ ID NOS:40-41) andTAP-2 (transporter associated with antigen processing). In mammals,genes encoding TAP-1, TAP-2 and two proteosome polypeptides are alllocated within the class II region of the MHC.

The class I pathway starts in the cytosol where proteins produced insidethe cell are degraded by the multicatalytic proteosome complex. Thepeptide products are translocated into the ER by the TAP proteins. Inthe lumen of the ER, the peptides bind the class I protein groove whilethe latter are complexed with the chaperone p88, □2m and TAP. Aftersecuring a peptide in its binding groove, the class I complex isreleased from TAP and transported through the Golgi apparatus to thecell surface. TAP genes are closely linked to the LMP2 (SEQ IDNOS:38-39) and LMP7 in the class II MHC gene cluster and belong to afamily of molecules involved in ATP-dependent membrane translocationknown as the ABC (ATP-binding cassette) transporters. TAP1 and TAP2function as a heterodimer each subunit having over 500 amino acids eachwith two hydrophobic domains, six membrane spanning regions and acytosolic ATP binding motif. Both TAP1 and TAP2 subunits are requiredfor peptide binding and translocation. TAP1 appears to be uniquelyinvolved in the interactions with class I/□₂ dimers at the luminalmembrane of the ER where it interacts with the membrane proximal regionof the a3 domain of class 1-□_(2m) complexes prior to peptide loading.Interaction between class I and TAP is crucial for efficient peptideloading. Antigen presentation is mediated by an additional factor,tapasin. TAP also binds 2M independently of class I heavy chain, perhapsfacilitating rapid assembly of class I peptide-binding complexes. TAPheterodimer may show a preference for amphipathic molecules as T cellantigenic determinants are often seen clustered around sequences whereamphipathic helical structures are predicted. TAP prefers peptides 8-10residues in length but may transport peptides ranging from 7-40residues..

Invariant chains are transmembrane glycoproteins found in intracellularcompartments in association with class II molecules. Multimersconsisting of three class I α□ dimers and three invariant chainsassemble rapidly in the ER and travel across Golgi bodies to thetrans-Golgi network that intersects with the endocytic pathway, whereclass II molecules reside for about 1-3 hr before transit to the cellsurface for display to T cells. Alternative splicing of the invarianttranscripts produces two isoforms p31 and p41 both of which can operateto assist folding of class II dimers, direct the passage of class IIfrom the ER through an exocytic pathway, and block loading of peptideuntil peptide sampling can occur as exocytic-endocytic pathwaysintersect. A four residue targeting signal at the N-terminus of theinvariant chain that is essential for intracellular transport toendosomal compartments. The C-terminus and the transmembrane region orthe invariant chain are also necessary for sorting of class II-invariantchain complexes to the endosome. p41 appears to regulate the productionof a stable 12-kDa SLIP-class II complex capable of enhancing SAgpresentation.

SAg-encoding nucleic acid is fused in frame with nucleic acid encoding aprotein involved in the antigen processing pathway such as the invariantchain or TAP which facilitates the expression of the SAg in the contextof MHC class I and II, respectively. Tumor cells, accessory cells andhybrids thereof are transfected with fused SAg-invariant chain DNA as inExamples 1 and 5. They are used in vivo as a preventative or therapeuticantitumor vaccine according to Examples 15, 16, 18-23. They are alsoused ex vivo for inducing tumor specific effector cells for adoptiveimmunotherapy of cancer (Examples 2-5, 7, 15, 16 18-23).)

SAg polypeptide post translationally is fused or associated withadditional molecules such as mono and diglycosylceramides, including butnot limited to -anomeric mono- and digalactosylceramides GalCer, α-Gal,glycosylated and prenylated SAgs. These constructs translocate with theappropriate trafficking molecule e.g., invariant chain, TAP, LMP, toselected surface receptor such as MHC class I, MHC class II or CD1.These transfectants are prepared as in Example 1. They are useful invivo as a preventative or therapeutic antitumor vaccine according toExamples 15, 16, 18-23. They are also useful ex vivo for inducing tumorspecific effector cells for adoptive immunotherapy of cancer (Examples2-5, 7, 15.16 18-23).

36. SAgs Combined with Signal Transduction Molecules or Heat ShockProteins (HSPs)

SAg-encoding nucleic acid is fused in frame to (or cotransfected with) anucleic acid encoding “signal transduction molecules” such as Ras, JAK 1and STAT-1A and heat shock proteins HSP-60, HSP-70, HSP-90a, HSP-90b,Cox-2 as well as heterotrimeric G proteins and ATPases. The genes forStaphylococcal HSP-70 (SEQ ID NOS:42-43) useful in this invention havebeen cloned (Ohta, T et al., J. Bacteriology 176: 4779-4783, (1994)). Asused herein, SAg polypeptides are ligated to any of above structures atthe peptide or nucleic acid level. Preferred proteins for thisembodiment are G proteins, ATPases and HSPs. Chemical conjugation iscarried out by conventional methods, e.g., use of preferredheterobifunctional crosslinkers. Alternatively, conjugates are producedgenetically as fusion proteins by conventional methods. In yet anotherembodiment, the conjugates are created by permitting natural binding ofthe components to each other without chemical modification. Any of theforegoing conjugates or fusion proteins may be used when incorporatedinto vesicles or exosomes secreted from a cell. See Example 36 formethods and protocols.

SAg-encoding nucleic acid is fused in frame (or cotransfected) withnucleic acid encoding a signal transduction protein or HSP.Transfectants are prepared as in Example 1. They are used in vivo as apreventative or therapeutic antitumor vaccine according to Examples 15,16, 18-23). They are also used ex vivo for inducing tumor specificeffector cells for adoptive immunotherapy of cancer (Examples 2-5, 7,15, 16 18-23).) The peptide or polypeptide conjugates are also usefulfor the same purposes.

37. SAgs with Specialized Sites for C-terminal GPI anchoring,Glycosylation, Sulfation, N-Myristoylation, Phosphorylation,Hydroxylation N-Methylation, Signal peptide binding, LPS binding, HSPbinding, Chemokine binding and Prenylation

SAg-encoding nucleic acid is fused in frame to nucleic acids encodingthe above “specialized sites” and transfected into tumor cells oraccessory cells The structures of these sites is given in Table 3, p. 48of Rocker R B I et al., J. Nutrition 123: 977-990 (1993).

Tumor or accessory cells express SAgs in a variety of fashions afterpost-translational modification (Wilkins, M R. et al., ProteomeResearch: New Frontiers in Functional Genomics Springer. Berlin, Germany(1997)). For example, myristoylated SAg will bind to surface lipids andwill be minimally secreted. In glycosylated form, the SAg will be routedto the class II pathway and appear bound on the cell surface. When boundto invariant chain, the SAg will be routed to the class II receptor.

Nucleic acids encoding proteins that active in post-translationalmodification of SAgs are fused in frame to nucleic acid encoding SAgs.These posttranslational modifiable sites include, but are not limitedto, a C-terminal GPI anchor, glycosylation site, palmitoylation site,myristoylation or prenylation site, N-methylation site, hydroxylationsite, phosphorylation site, sulfation site, signal peptidase site,carboxylation site and prenylation sites.

The incorporation of many membrane proteins into the lipid environmentis based on sequences of largely hydrophobic amino acids that can formmembrane spanning domains. However, a large number of membraneassociated proteins do not display hydrophobic elements in their primarysequences. The capacity for membrane association in these cases is oftenprovided by covalent attachment (either cotranslationally or posttranslationally) of lipid groups to the polypeptide chain. Acylation ofproteins by addition of C14 myristic acid to an N-terminal gly residueor addition of C16 palmitic acid by thioester linkage to cysteineresidues is in a variety of positions in SAgs. Palmitoylation of SAgs isnot restricted to thioester linkage and may occur through oxyesterlinkages to serine and threonine residues. Furthermore, thioesterlinkage of fatty acyl groups to proteins is not restricted to palmitate.Longer chain fatty acids such as stearic acid (C18) and arachidonic acid(C20) are also produced. The addition of palmitoyl and/or myristoylgroups with varying lengths confers additional and sufficient bindingenergy for hydrophobic binding of proteins to receptors, membranes orlipid bilayers. The attachment of palmitate is sufficient whereas theattachment of myristate is insufficient in isolation. Palmitoylationthus provides a means for membrane anchorage of SAgs and can alloweffective concentration of an enzyme or other regulatory proteins at themembrane.

Glycosylated SAg is better capable of binding to oligosaccharidereceptors on blood vessels, inflammatory cells or immunocytes. Signalpeptide sequences permit the SAg to be routed to various cell surfacereceptors. Prenylation is important in the membrane attachment andprotein-protein interactions of SAgs and oncogene activation.Prenylation, or post translational enzymatic addition of prenyl,geranyl, famesyl or geranylgeranyl, involves reactions of a prenyldiphosphate with a cysteinyl sulfhydryl group near the C terminus of theprotein to give a prenyl-S-Cys moiety. Characteristically theCys-ali-ali-Xaa sequence (“ali” is an aliphatic amino acid; Xaa is anyamino acid) is recognized by the transferase that catalyzes thereaction. When Xaa is serine, alanine or methionine, the protein isfamesylated; when Xaa is leucine, it is geranylgeranylated.Farnesylation of the protooncogene p21^(ras) is integral both for itsmembrane association and transforming activity. Farnesylated proteinsmediate the induction by IL-1□ of NOS whereas a geranylgeranylatedproteins repress this induction.

Nucleic acids encoding HSPs, along with their promoters, are fusedin-frame (or cotransfected) with SAg nucleic acid. These include but arenot limited to two recently discovered HSP genes, orf37 and orf 35 inStaphylococcus aureus that are upstream and downstream of grpE(hsp20),dnaK(hsp70) and dnaJ(hsp40) in the following sequence:orf37--hsp20--hsp70--hsp40--orf35. The promoters are located upstream oforf37 and upstream of hsp40. These fused proteins are useful aspreventative or therapeutic antitumor vaccines according to Examples 15,16, 18-23. They are also useful ex vivo for producing a population ofanti-tumor T cells, NKT cells or NK cells for adoptive immunotherapy ofcancer (Examples 2-5, 7, 15, 16, 18-23).

Most eukaryotic cells are decorated with chemical groups such asphosphates, methyls, sugars, or lipids during or after their translationfrom mRNA. These extra groups have various functions, often serving asswitches or localization signals. One lipid modification is proteinprenylation in which a 15-carbon farnesyl or 20-carbon geranylgeranylgroup is attached to the protein's —COOH terminus followed by othermodifications (proteolysis, methylation, and palmitoylation).

Most prenylated proteins are members of signal transduction cascades.For example, the -subunits of heterotrimeric guanosine triphosphate(GTP)-binding proteins (G proteins) and virtually all members of the Rassuperfamily of proteins. Farnesylation of H, K, N-Ras is essential forthe ability of oncogenic mutants of these proteins to transform cells.30% of established tumor cell lines contain mutationally activated Rasproteins. FTase inhibitors shrink tumors in animals to an undetectablesize with no significant toxicity after weeks or months of exposure.Farnesylation is a prerequisite for palrnitoylation. Palmitoylation ofH-Ras occurs only in the plasma membrane by a putative membrane-boundpalmitoyl transferase. Farnesylation may bring a finite amount of H-Rasto all cell membranes, at which point and palmitoylation is required totrap it in the plasma membrane. H-Ras palmitoylation like Gprotein-subunit palmitoylation, is reversible and may regulate signaltransduction. COOH terminal proteolysis of prenylated proteins andmethylation are required for palmitoylation, membrane binding and Rasfunction. Prenyl protein specific protease and methyltransferase likeFtase may be good targets for drugs that prevent oncogenesis.

Common N terminal additions are fatty acid acylations and glycosylationswhich provide polypeptide chains with short “lipophilic handles” orrecognition sites that serve to facilitate their vectoral transport orcompartmentalization are common N-terminal additions. For example,myristic acid in the form of myristyl CoA serves as a substrate forspecific N-terminal acylations that are important in anchoring proteinsto endoplasmic membranes. The most common C-terminal modifications areamidations, acylations, polyadenylations and the enzymatic additions oftyrosyl residues. Similarly the C-terminal acylation process is complex.Prenylation occurs at Cys residues is often associated with proteinsthat end in Cys-Val-Ile-Ala. The reaction sequence involves (1) a firstprenylation (addition of a farnesyl moiety to Cys) followed by (2)cleavage of the Ala, Ile and Val residues and (3) the carboxymethylationof the resulting C-terminal prenylated cysteine. In addition toproviding a membrane anchor, this modification often is essential tofunction of oncogenes such as Ras.

Two separate and well characterized pathways for carbohydrate addition:the N-linked dolichol pyrophosphate mediated pathways and the O-linkedpathways that utilize UDP sugars as substrates and hydroxylated aminoacid side chains as sites for attachments. Side chains aminophosphorylation of specified proteins usually at tyrosyl or serinylresidues as a way of causing cascade-like amplifications in a metabolicsystem. Methylation and methyl additions can also serve as novel on-offswitches for metabolic processes. The targeted amino acids or methyladditions are lysine, histidine and arginine. In prokaryotes, reversiblemethylations of aspartyl and glutamyl side chains can occur. The bestexample is carboxymethylation of glutamate which is associated withbacterial chemotaxis and is elaborated by the opening and closing ofmembrane ion channels upon methylation and demethylation. Posttranslational modifications can lead to crosslinking and stabilizationof protein matrices. Amino acids such as L-lysine, L-glutamine,L-cysteine and L-tyrosine are utilized extensively as sources forprotein cross-linking. Examples include the extracellular matrix crosslinking of collagen and elastin and the stabilization of keratin-derivedmatrices and tubulin by -glutamyl lysine crosslinks.

In bacteria the majority of proteins that form durable wall associationspossess either distinctive N-terminal signals (lipoproteins) or morecommonly distinctive C terminal wall associating signals although anumber of wall associated proteins possess neither of these types ofsignals. A number of wall-associated proteins in gram-positive bacteriaare anchored to the external surface of the cytoplasmic membrane via acovalently attached lipid moiety. Both gram-negative and gram-positivelipoproteins possess similar distinctive N-terminal signal sequenceswhich contain a tetrapeptide consensus at the cleavage site consistingof Leu-X-Y-Cys where X and Y are predominantly small neutral residuesand signal and signal peptidase cleavage occurs between Y and Cys. Thissequence directs either co- or post translational modificationsinvolving transfer of glycerol from phosphatidylglycerol to the +1 Cys,followed by the transfer of fatty acids from phospholipid to theglyceryl-prelipoprotein to produce a diglyceride-prelipoprotein. The Cterminal end of a large number of Gram positive wall-associated proteinsshare common structural features that are required to localize theseproteins in the cells wall. These C-terminal structures include a numberof distinct features. At the extreme C-terminus there is a stretch of15-22 hydrophobic residues, followed by a short tail of predominantlycharged amino acids. Immediately upstream from thishydrophobic/charged-tail domain, there is a highly conservedLeu-Pro-X-Thr-Gly-X (LPXTGX) motif which is usually preceded by asequence containing a high proportion of regularly spaced prolines. GPIanchors have not been identified on bacterial cell surface proteins. Butthe strong conservation of the LPXTGX motifs and of ahydrophobic/charged tail residue-helical domain are common structuralfeatures that are required to localize these proteins in the cell.Protein A is covalently coupled to the cell wall whereas of the proteinsare not. Non-covalent interactions may occur in some proteins holding itin the cell wall while cross-linking occurs around proline rich regionto form peptidoglycans. Hydrogen or water binding sites can be createdby hydroxylation reactions, e.g., hydroxylation of proline in collagenprovides sites for intrachain hydrogen and H₂O bonding.

SAg-encoding nucleic acid is transfected into cells together with codingregions to permit the above post translational modifications whichcontribute to the production of an immunogenic tumor cell accessory cell(preferably a DC) or a tumor cell/accessory cell hybrid. Such nucleicacids encoding the sites for post-translational modifications of SAgsare useful in the structural modification, translocation, cell surfacebinding and association with key energy-producing and signaltransduction molecules and receptors. The cells expressing the productsof these post-translational modifications are useful as a preventativeor therapeutic antitumor vaccine according to Examples 15, 16, 18-23).They are also useful ex vivo for producing a population of anti-tumor Tcells, NKT cells or NK cell for adoptive immunotherapy of cancer(Examples 2-5, 7, 15, 16, 18-23).

38. SAgs and SAg Proteomes for Enhanced Immunogenicity, Specificity andIntracellular Trafficking of Soluble or Cell-Bound Binary or TernaryComplexes

SAgs with genetically engineered binding sites are provided in order toenhance their coupling to bioreactive complexes, peptides and LPS's andgalactosylceramides. SAgs with a glycosylation other glycosylceramidebinding site bind to glycosylceramide-CD1 or glycosylceramide-CD1complexes alone in soluble or immobilized form, or cell bound afterbinding to a receptor on a T cell or NKT cell. SAgs are also providedwith an LPS binding site for binding to soluble, immobilized or cellbound LPS-CD14 complexes.

SAgs are provided with a glycosphingolipid or glycosylceramide site bywhich they can bind to CD1-glycosylceramide or CD1-glycopsphingolipidcomplexes present in soluble, immobilized form or affixed to CD1+ cellsor NKT cells. Glycosylated SAgs are bound to CD1-glycosylceramidecomplexes in soluble form or fixed to CD1+ cells or NKT cells. SAgs arealso provided with an overexpressed site for MHC class I molecules, toincrease the effectiveness of binding to MHC class I-tumor peptideantigen complexes or TCR-bound MHC class I-tumor peptide complexes.

SAgs are engineered with repeating peptides which bind to the V□ chainto increase clustering. SAgs with an “overexpressed” (in terms ofnumber) SAg receptor site binds to tumor cells expressing SAg receptors.SAgs possess a site for binding HSPs which are useful in immunizingnormal or anergic T cells in a tumor patient. SAgs bind to T cellantagonist MHC-tumor peptide complexes converting the binary complex toa ternary complex with T cell agonist activity. Anergic T cells areactivated by these ternary complexes.

SAgs are prepared with an overexpressed site for bindingglycosphingolipids or glycosylceramides. These complexes are loaded ontoCD1 receptors of antigen presenting cells and presented to the tumorbearing host either in vivo or ex vivo (Examples 4, 5, 7). SAgs with amyristoylation site will bind to bacterial glycolipids such aslipoarabinan or a mycolic acids The binary complex is then loaded ontoAPCs expressing CD1 receptors. These cells are then used in vivo(Example 14, 15, 16, 18-23) to produce a tumoricidal response.Alternatively, they are used ex vivo to produce tumor specific effectorT or NKT cells for adoptive immunotherapy (Examples 2, 7, 14, 15, 16,18-23).

A SAgs may also be prepared with signal sequences for protein sortingand intracellular trafficking. Signal sequences comprise short stretchesof amino acids located at the N terminus of a protein, the C terminus orin the middle of the peptide chain. The physical properties of thesesequences e.g., their polarity or charge. Signal regions are threedimensional domains on the surface of a protein made up of differentfragments of the same peptide chain or by different chains altogether.Structural signals are recognized and bound by receptors located on themembranes of organelles. Signal sequences also serve as recognitionsites for enzymes which modify the proteins altering their propertiesand bring about a change in their fate. Once they have fulfilled theirfunction, some of the signal sequences are removed by sequence specifichydrolases. Signal peptides fused to SAgs guide them to the secretory orexocytosis pathway, or to proteins localized to the endoplasmicreticulum, lysosomes, mitochondria, nucleus, peroxisomes or secretoryvesicles.

GPI-SAg-Ceramide or GPI-SAg-CD1-Ceramide Complexes Expressed on TumorCells, Antigen Presenting Cells or APC/tc Hybrid and Shed as Exosomes

Cells expressing, overexpressing or shedding GPI proteins are preparedso that they comprise covalently- or noncovalently bound mono- ordiglycosylceramides with terminal or subterminal α1-2, α1-4 or α1-6configurations and SAg protein or peptide moieties.

The synthetic pathway involves transfection of SAg DNA into a tumor cellor accessory cell or a hybrid thereof The SAg protein is translated in aprecursor form consisting of a receptor-coding region sandwiched betweenamino and carboxy-terminal sequence signals. In the endoplasmicreticulum, the signal peptides are cleaved and a GPI anchor comprising aglycosylceramide optionally bonded to a phytosphingosine chain isattached at a specific site designated ω. Further post-translationalmodifications are made in the Golgi before trafficking to the outerleaflet of the plasma membrane. Once GPI-SAg molecules arrives at thecell surface, they may remain entirely mobile within the lipid bilayeror may associate within membrane subdomains.

GPI-SAgs are released from the cell surface into the extracellularmilieu. They leave the cell surface as SAg-glycan-lipid complexes, asSAg-glycan complexes or as free SAgs devoid of a GPI anchor. GPI-SAgsreleased from intact cell are also released free of their lipid moiety,hence their designation as LIP(−) GPI-SAgs, whereas those presumablyreleased with an intact lipid moiety are termed LIP(+) GPI-SAgs. Thelipid free moieties are more hydrophilic and therefore soluble in anaqueous environment, whereas the intact lipid-glycan-protein complexestravel in more hydrophobic environments. In the absence of detergents,the released or “shed” LIP(+)-GPI-SAgs in vivo are vesicles with clearlydefmed lipid bilayers or as hydrophobic aggregates lacking a bilayermorphology. These shed vesicles, often referred to as exosomes, containmany LIP(+) GPI-SAgs. The shedding process itself appears to depend onGPI-proteins, because vesiculation is reduced by 50-90% in cells lackingGPI proteins. Shedding is enhanced by treating the tumor cells with 20μM retinoic acid. In addition high concentrations of glycosphingolipidson the tumor cell surface are generated by selective transport from thesite of synthesis to the cell surface. Provision of ceramide containingthe α2-hydroxy fatty acid C₆OH results in (1) conversion togalactosylceramide, galabiosylceramide and sulfatide and (2) sorting inthe trans-Golgi network to the tumor cell surface. GPI-SAgs remainbiologically active after being released from the outer leaflet of cellmembranes. LIP(+) GPI proteins may also transit to adjacent membraneswhere they associate with the exogenous membranes by incorporatingthemselves into the lipid bilayer in addition to binding to surfacereceptors.

Additionally, superantigen or oxyLDL receptor nucleic acids aretransfected into yeast sec mutant. The yeast sec mutant, 6-4, contains atemperature senstive mutation in a gene product required for thetransport of secetory vesicles for the trans-Golgi network to the plasmamembrane. Gene expression is initiated by an inducible promoterconcomitant results in the arrest of vesicle fusion and the insertion ofSAg or LDL receptor protein in the plasma membrane. Thus gene expressionbegins at the same time that secretory vesicles become unable to fusewith the plasma membrane, ensuring that the desired gene productsaccumulate in the membranes of these vesicles. The purification of thesevesicles is rapid and simple, thereby facilitating the subsequentcharacterization of the desired gene product. Because the Sec6 proteinis known to be involved only in the fusion of these vesicles with theplasma membrane, translocation and processing of proteins in theendoplasmic reticulum and processing in the Golgi are largley unaffectedby the Sec6 mutation. The transfected superantigen or LDL nucleic acid(plasmid) is expressed as superantigen polypeptide or oxyLDL polypeptidein vesicles in association with yeast GPI-lipid membrane structures. Thelipid portion of the SAg-GPI-lipid complex comprises a ceramide with aC26 dihydroxy sphingosine or phytosphingosine configuration which isessential for activating NKT cells. The resultingSAg-GPI-phytosphingosine vesicles have the capacity to activate T cellsvia the superantigen and NKT cells via the phytosphingosine and thusproduce a potent anti-tumor effect. Administered preferably by directadministration into the tumor the oxyLDL receptors induce an excessiveaccumuation of endogenous or exogenously administered oxyLDL and LDL atthe tumor site. The deposited oxyLDL induces apoptosis and foam cellformation in tumor cells and tumor microvascular enodthelial cellsresulting in potent tumoricidal response. Optionally,SAg-GPI-phytosphingosine are expressed on these vesicles together withvesicles expressing or oxyLDL receptor-GPI-phytosphingosine oxy LDLreceptors

Vesicles containing SAg-GPI-phytosphingosine or oxyLDLreceptor-GPI-phytosphingosine are prepared and isolated according themethod of Coury L A et al., Methods in Enzymology 306: 169-186 (1999)and as in Examples 4, 5, 7, 42, 50-51. They are useful in vivo as apreventative or therapeutic antitumor vaccine according to Examples 14,15, 16, 18-23, 36 They are also useful ex vivo for producing apopulation of tumor specific effector T or NKT cells for adoptiveimmunotherapy of cancer (Examples 2-5, 7, 15, 16, 18-23).

39. Effector T Cells: Methods of Lowering Activation Threshold forActivation by SAg

Tumor peptide MHC complexes are insufficient to activate T or NKT andmay even induce antagonism or anergy. SAgs added to the complexes areuseful to overcome activating T or NKT cells and overcoming the anergycommon in tumor-bearing hosts. To enhance responsiveness totumor-peptide-MHC-SAg complexes and to overcome anergy, it is desirableto reduce the threshold for signal transduction in an effector T or NKTcell population. To accomplish this, nucleic acids encoding SAg-specificTCR V□ regions are transfected into T or NKT cells to duplicate orotherwise induce overexpression. In addition, measures are taken toalter signal transduction by dimerizing the tyrosine kinase receptors ordeleting the inhibitory region of the TCR.

Most SAgs show selective binding to well defined segments of the V□chain of the TCR. The TCR genes are clustered on chromosome 7 andinclude 75-100 V, 2D, 13 J, and 2 C□ genes. The entire 685-kb humanlocus has been sequenced, the longest contiguous subfamilies thatexhibit >75% sequence identity at the DNA level. The human TCR locus ison chromosome 14 and consists of 42 V genes, 61 J genes and 1 C□ gene.The TCR chain genes are on chromosome 6 and consist of approximately23V, 2D, 12J, and 2C gene segments. The 2 C□ genes form clusters withupstream D□ and J□ segments: C□1 rearranges only with D□1/J□1 geneswhereas C□B2 rearranges with both D□ and J□ segments. Similarly,functional V□ genes appear to rearrange to both J clusters in a randomfashion. The □ chain transcripts of antigen-specific T cell clonesappear to contain little length variation and harbor conserved Nadditions.

A mechanism for achieving diversity in variable (antigen-specific)regions of the TCR involves the random addition of nucleotides insertedat junctional positions during the joining of V□D□J segments. It is atthis position that nucleic acids encoding the major V□ binding site fora specific SAgs are inserted. This overexpression allows for moreselective recognition of SAg and a lower activation threshold by a SAgthat selectively binds at that site.

Nucleic acid encoding SAg receptor is amplified and transfected into Tcells to overexpress the SAg receptor on the cell surface These T cellsbind SAgs, and this is linked to appropriate signal transductionpathways that deliver a mitogenic signal to the T cell. One method ofincreasing T or NKT cell reactivity to a SAg is to increase the densityof their SAg receptors. Even in the absence of ligand, the equilibriumis shifted from monomeric inactive receptors to dimeric or oligomericactive receptors. Concomitant expression of the corresponding ligandreinforces the signal. Increased numbers of receptors occur afterincreased transcriptional activation of, or amplification of, the SAgreceptor gene. Amplification is the preferred method.

The SAg receptor may also be mutated so that it engages inligand-independent dimerization. Examples of such mutations are additionor loss of a cysteine residue in the extracellular domain causingformation of dimeric and disulfide bonded and activated receptors. Inaddition it is possible to dimerize tyrosine kinases by fusing atyrosine kinase catalytic domain to a protein which is a functionaldimer. These fusion partners are able to form homodimers. Such a fusionprotein results in dimerization of kinase domains which allows theirautophosphorylation and activation. Interaction with receptors in amanner which promotes dimerization of two different receptors is anothermethod to enhance receptor reactivity. The kinase domain of a receptormay be mutated to increase catalytic activity or alter substratespecificity. Such mutations expand quantitatively and qualitatively therepertoires of substrates in the target cells and thereby shift thebalance towards activation and transformation. Mutations in regionsinvolved in negative regulation of receptor function also contribute tothe transforming properties. Loss of regions in the C terminus that areregulatory serine phosphorylation or autophosphorylation sites alsocontributes to excessive receptor activity.

Effector cells as discussed above are prepared as in Examples 4, 5, 7.They may also be used in vivo as tumor specific effector (T or NKT)cells for the adoptive immunotherapy of cancer (Examples 2-5, 7, 15, 16,18-23).

40. SAg Nucleic Acids Fused of Cotransfected into Tumor Cell withNucleic Acids Encoding Inducible Nitric Oxide Synthase (iNOS)

SAg-encoding nucleic acid is fused in frame (or cotransfected) withnucleic acid encoding inducible nitric oxide synthase which producesnitric oxide (NO). NO is derived from terminal guanido-nitrogen ofL-arginine which is catalyzed by the constitutive or inducible nitricoxide synthase (iNOS). NO is pleiotropic and is a major cytotoxicmediator secreted by activated endothelial cells and macrophages.Production of NO is associated with apoptosis of tumorigenic cells andwith a bystander effect on surrounding non-NO producing tumor cells(bystander effect). Non metastatic tumor cells show high levels of iNOSactivity and NO, whereas metastatic cells do not. There is an inverserelation between production of endogenous NO and the tumor cellssurvivability. In the present invention, tumor cells transfected withSAg-encoding nucleic acid are cotransfected with nucleic acids encodingiNOS. The gene for iNOS has been cloned and characterized by Xie Q etal., Science 256: 225-228 (1992). Tumor cells cotransfected with nucleicacids encoding SAgs and iNOS demonstrate augmented immunogenicity viathe expression of SAg as well as enhanced auto- and bystandertumoricidal capacity via NO production.

After administration to a patient and colonization of metastatic sites,the transfectants induce a powerful local and systemic tumoricidaleffect. The presence of NO allows the transfectants to die naturally viaauto-apoptosis within a finite period (usually 72 hours) afteradministration thus minimizing the risk of inducing active metastaticdisease. These tumor cell transfectants may also be made to expressoncogenes associated with the metastatic phenotype to promotelocalization of the cells to tumor sites in vivo. The cells may befurther transformed by nucleic acid encoding angiostatin or otherangiogenesis inhibitors for additional tumoricidal potency. Thetransfectant are prepared by methods in Example 1-3 and used as apreventative or therapeutic antitumor vaccine by methods in Example 15,16, 18-23).

41. DCs, Other Accessory Cells and DC/tc Hybrids Expressing and/orSecreting SAg

Accessory' cells are necessary to generate primary antibody responses inculture. Of the various types of accessory cells, DCs are the mosteffective APC. DCs are a preferred accessory cell. However, theinvention is not confined to DCs. Any other accessory cell type may beused in place of DCs. In particular, accessory cells are defined inOxford's Dictionary of Biochemistry and Molecular Biology as includingfibroblasts, synoviocytes, macrophages, B cells, Langerhans cells andany other cell type which assists in producing an immune response of anykind.

DCs have exceptional capability to capture antigens, process and presentantigenic peptides, migrate to lymphoid organs, and induce primaryimmune responses of both CD8+ and CD4+ T cells. The ability of DCs toact as potent APC in the induction of T cell responses is attributed tothe high expression of MHC molecules and adhesion and/or costimulatorymolecules as well as the cells' capacity for to producing cytokinesessential for the activation and proliferation of the T cells.

The number of molecules of antigen-MHC complex on tumor (and infected)cells is typically small (100 per cell), and are recognized by rareT-cell clones (at a frequency 1/100,000) via a TCR that has a lowaffinity (1 M). In vitro or in vivo, only a few DCs are necessary toprovoke a strong T-cell response. In the mixed leukocyte reaction, oneDC was sufficient to stimulate 100-3,000 T cells. MHC products andMHC-peptide complexes are 10-100 times higher on DCs than on other APCssuch as B cells and monocytes. Mature DCs resist the suppressive effectof IL-10, but synthesize high levels of IL-12 that enhances both innate(NK cell) and acquired (B and T cell) immunity. DCs also express manyaccessory molecules that interact with various molecules or receptors onT cells to enhance adhesion and signalling (co-stimulation): examples ofsuch pairs are LFA-3/CD58, ICAM-1/CD54, B7-2/CD86. Tumor cells thatexpress the B7 gene elicit CTLs against otherwise silent, subdominanttumor antigens. All these properties of DCs (MHC expression, CD1expression, secretion of IL-12 and the expression of co-stimulatorymolecules) are upregulated within a day of exposure to many stresses and“dangers” including microbial products.

Infected cells and tumors frequently lack the costimulatory moleculesthat drive clonal expansion of T cells, the production of cytokines, andT cell development into killer cells. Located in most tissues, DCsovercome challenges by capturing and processing antigens, and displayinglarge amounts of MHC-peptide complexes on their surface. They upregulatetheir co-stimulatory molecules and migrate to lymphoid organs, thespleen and draining lymph nodes, where they activate antigen-specific Tcells. All of these activities of DCs can be induced by infectiousagents and inflammatory products, so that DCs appear to function as“mobile sentinels” that not only bring antigens to T cells but alsostimulate those T cells in the induction of immunity.

DCs are present in most tissues in a so-called “immature” state, unableto stimulate T cells. Although these DCs lack the requisite accessorysignals for T-cell activation, such as CD40, CD54 and CD86, they arewell equipped to capture antigens, a key event in the induction ofimmunity; the antigen is then able to induce full maturation andmobilization of the DCs. Terminally-differentiated or mature DCs canreadily prime T cells Once activated by DCs, these T cells can completethe immune response by interacting with B cells for antibody formation,macrophages for cytokine release, and target cells resulting in lysis.Thus, immature DCs first handle antigens and then, as mature DCs a dayor more later, they potently stimulate T cells.

DCs stimulate CTLs, which express the accessory molecule CD8 andinteract with MHC class I bearing cells, to proliferate vigorously. Inthe presence of mature DCs and of IL-12, CD4-expressing T-helper cellsturn into interferon gamma (IFN)-producing TH-1 cells. IFN activates theantimicrobial activities of macrophages and, together with IL-12,promotes the differentiation of T cells into killer cells (CTL). Thecapacity of DCs to produce IL-12 and stimulate TH-1 cells leads tomicrobial resistance. Through IL-4, DCs induce T cells to differentiateinto TH-2 cells which secrete IL-5 and IL-4, activate eosinophils andhelp B cells generate an antibody response, respectively. DCs respond toT cells as well. CD40 and the newly described TRANCE/RANK receptor onDCs are ligated by the TNF (tumor-necrosis factor) family of proteinsexpressed on activated and memory T cells; this leads to increased DCsurvival and, in the case of CD40, upregulation of CD80 and CD86,secretion of IL-2 and release of chemokines such as IL-8 and MIP-1α and□.

Immature DCs capture antigen (and particles and microbes in general) byphagocytosis. They then form large pinocytic vesicles in whichextracellular fluid and solutes are sampled, a process calledmacropinocytosis. Finally, they express receptors that mediateadsorptive endocytosis, including C-type lectin receptors like themacrophage mannose receptor and DEC-205, as well as Fc, located in mosttissues, and Fc receptors. Macropinocytosis and receptor-mediatedantigen uptake make antigen presentation so efficient that picomolar andnanomolar concentrations of antigen suffice, much less than thenicromolar levels typically employed by other APCs. However, once the DChas captured an antigen, which also provides signals to mature, itsability to capture antigens rapidly declines, and the cell begins toassemble antigen-MHC class II complexes.

An antigen enters the endocytic pathway of the DC. DCs produce largeamounts of MHC class II-peptide complexes due to specialized, MHC classII-rich compartments (MIICs) that abound in immature DCs. MIICs arelate-endosomal structures that contain the HLA-DM or H-2M products,which enhance and perform editing functions in the binding of peptide toMHC class II. During maturation of DCs, MIICs convert to non-lysosomalvesicles that discharge their MHC peptide complexes to the surface.

To generate cytotoxic killer cells, able to eliminate infected cells,and attack tumor cells and transplanted foreign cells, DCs must presentpeptides (complexed generally to MHC class I proteins) to CD8+ T cells.Display of peptide-loaded MHC class I complexes on the DC surfacefollows translocation by a peptide transporter from the cytosol to theER, where complexing occurs and then to the surface.

Human DCs are characterized by a pattern of surface markers and have thephenotype CD1a+, CD3^(neg), CD4^(neg), CD8^(neg), CD20^(neg), CD40+CD86+ in the human. The murine phenotype is and CD3^(neg) CD4^(neg),CD28^(neg), CD8- B220^(neg), CD40+, CD80+ and CD36+.

Maturation of DCs is required for the initiation of an immune response.Microbial products including whole bacteria and the bacterial cell-wallcomponent LPS and inflammatory mediators such as IL-1, GM-CSF and TNF,stimulate DC maturation, whereas IL10 blocks it. Ceramide, which isinduced by maturation signals, shuts down antigen uptake by the DC.Mature DCs express high levels of the NFKB family of transcriptionalcontrol proteins (RelA/p65, RelB, RelC, p50, p52) which regulate theexpression of many gene encoding immune and inflammatory proteins.Signalling through the TNF-receptor family, for example TNF-R(CD-120α/□), CD40, and TRANCE/RANK, results in activation of NFκB.Therefore, to induce an immune response through activation of DCs, apathogen or antigen may have to mobilize the signal transductionpathways of the TNF-R family and TNF-R-associated factors (TRAFs).

One explanation for the failure of the immune system to eradicate mostimmunogenic tumors is the lack of tumor antigen presentation by DCs invivo. Several strategies using tumor antigen-charged DCs as vaccines forcancer immunotherapy have been developed. Immunization with DCs pulsedwith purified tumor-associated peptides or proteins has been shown to bea powerful method of priming tumor-reactive T cells and inducing hostprotective and therapeutic antitumor immunity in mice and man. However,such a clinical approach is currently limited due to the paucity ofidentified human tumor rejection antigens. The polymorphism of the HLAsystem has also made it difficult to identify tumor-associated peptidesas cancer vaccines. In human melanoma, a class of tumor-associatedproteins has been identified. However, it is unclear which antigen isthe best choice for effective tumor rejection in vivo or how effectiveany such antigen may be. Thus, immunization with defmed tumor antigensis currently limited to a small number of cancers in which candidateantigens have been identified. Anichini et al., J. Immunol. 156:208-217(1996), showed that the majority of CTL present in HLA-A2. 1+ melanomapatients were not directed to the known tumor antigens, Melan-A/Mart-1,tyrosinase, gp100 or MAGE-3. Therefore, immunization with other, yetunidentified, antigens would be more effective in eliciting tumorimmunity in these patients. Johnston et al., J. Exp. Med. 183:791-800(1996) demonstrated that the enhanced immunogenicity of tumor cellsengineered to express the B7-1 gene was a result of expansion of theantigenic repertoire of the tumor. This implies that vaccination withmultiple tumor antigens may be superior to use of a single dominantepitope. Indeed, in situations where a tumor-associated antigen remainsunidentified, a novel approach is needed for presentation of thatantigen by a professional APC.

An alternative approach, not encumbered by these limitations, is to useunfractionated tumor peptides or tumor proteins as a source of tumorantigens. Two studies have shown that administration to mice of APC(from the spleen) or epidermal Langerhans cells pulsed with tumorfragments resulted in protective immunity against tumor challenge.Zitvogel et al., J. Exp. Med. 183:87-97 (1996) showed that vaccinationof mice with bone marrow-derived DC pulsed with unfractionated tumorpeptides reduced the growth of subcutaneously established, weaklyimmunogenic tumors. Thus, immunization with multiple tumor antigens maybe superior to use of a single dominant epitope.

One approach to overcome the possible drawbacks of unfractionated tumorantigens is to use mRNA from tumor cells as a “source” of antigen. mRNAcan be amplified from a very small number of cells, permitting thegeneration of sufficient amounts of antigen from minute amounts of tumortissue Moreover, tumor-specific mRNA can be enriched by subtractivehybridization to remove RNA that is common to normal tissue. Thisincreases the levels of the relevant tumor-specific antigen(s) that canbe achieved, and hence, the potency of the vaccine. More importantly,this approach reduces the concentration of nonspecific antigens or,possibly, self-antigens, thereby lessening the potential forautoimmunity. Pulsing DCs with RNA is known to be effective inempowering them to induce CTL responses and tumor immunity.

The fusion of tumor cells with DCs is another approach to generate ahybrid vaccine that has both potent antigen processing/presenting poweralong with the endogenous expression of multiple tumor antigens. Such ahybrid cell would be more effective in inducing antitumor immunity. Gonget al., Proc. Natl. Acad. Sci U.S.A 26:6279-6283 (1998), demonstratedthat fusion of a relatively immunogenic mouse tumor, MC38 carcinoma,with syngeneic DCs resulted in a vaccine that induced (1) T cellprotective immunity against tumor challenge and (2) rejection of anestablished tumor. Wang et al., J. Immunol. 161:5516-5524 (1998) usedthe poorly immunogenic B16 (B16.F10) melanoma which does not express MHCand costimulatory molecules. Immunization with irradiated B16 tumorcells failed to induce systemic immunity or elicit functionaltumor-reactive T cells. RMA-S is a Rauscher MuLV-induced T cell lymphomaoriginating in a C57BL/6 (“B6”) mouse that is genetically defective inTAP, and thus, does not process endogenous antigens for binding to MHC.Fusion of DCs with syngeneic tumor cells generated hybrid cells thatexpressed both DC-associated accessory molecules important for antigenpresentation and tumor-derived antigens. The DC/tc hybrids wereprocessed and presented tumor-associated antigens and elicitedtumor-reactive CTLs. Vaccination of B6 mice with B16/DC hybrid cellsinduced partial protective immunity against tumor challenge.Immunization with B16/DC or RMA-S/DC hybrid cell vaccines primed lymphnode (LN) T cells, which, after expansion ex vivo, were active inadoptive immunotherapy. The transfer of such vaccine-primed, expanded Tcells into tumor-bearing mice reduced the number of established B16pulmonary metastases and, in the case of RMA-S/DC, effectivelyeradicated disseminated FBL-3 tumor.

The present invention includes a hybrid cell made from fusion of a tumorcell and a DC cell further transformed or transfected with a SAg.Nucleic acids encoding SAgs may be introduced into either the tumorcells or the DCs prior to fusion as in Example 1, 2, 3, 25, 26. Thisfused cells are prepared as in Example 24, 25 and their phenotypeestablished by the retention of DC characteristics, tumor cell antigensand the expression of SAg (Example 25). By virtue of these multiplefeatures, this SAg-expressing DC/tc has the unique capacity activatemaximally an anti-tumor immune response.

SAg stimulation is known to activate CD4+ and CD8+ T cells to recognizeand lyse tumors specifically both in vitro and in vivo. The DC componentof the hybrid cell provides optimal tumor antigen presentation due toits enormous surface area together with natural expression ofcostimulatory molecules B7.1, B7,2, adhesion molecules ICAM-1 andICAM-3, MHC class I and class II and CD1 receptors. B7.1, in particular,provides a basis for expanding the epitope recognition spectrum fromdominant to subdominant epitopes. The expressed SAg confers upon thehybrid cell an augmented capacity to activate various classes of cellsthat mediate both innate and “acquired” or adaptive immunity, includingCD4+ and CD8+ T cells, NK cells and NKT. The SAg also contributes togeneration of TH-1 cytokines by this class of T helper cells whichcontributes to an optimal anti-tumor response. The DC/tc hybrid thatexpresses and/or secretes SAg is abbreviated herein as an “S/D/t” celland combines the potent activating properties of SAg with thespecialized (tumor) antigen presenting capacity of the DC and the tumorantigens provided endogenously by the tumor cell partner. This S/D/tcell thus consolidates in a single cell the capacity to unleash andamplify the full weight of the host immune response specifically againsta selected array of tumor associated antigens.

The present invention also includes the additional introduction, intothe S/D/t cell of with additional nucleic acids. In one embodiment, theadditional nucleic acid encodes the particular galactosyltransferaseenzyme that catalyze the synthesis of the “heterograft epitope” Gal. Inanother embodiment, the additional nucleic acid encodes enzymes thatsynthesize galactosylceramide which is the “natural” epitope recognizedby the invariant chain of NKT cells.

To summarize the foregoing section, the present invention includes DCs,other accessory cells or hybrid DC/tc, each transformed to express SAgsas described in Examples 1 and 3. The transformed (or transfected)hybrid cell, the S/D/t cell, expresses (1) the major accessory moleculesof DCs cells (such as CD40, CD80 and CD86, MHC class I and II and CD1);(2) tumor associated epitopes provided by the tumor cell fusion partner;and (3) SAg either membrane bound, secreted or both which activates Tcells, NK cells and NKT cells to produce a specific or selectivetumoricidal response.

While the tumor S/D/t cells are preferred, SAg-transfected DCs or otheraccessory cells are also effective in inducing antitumor responses.These are used as a preventative or therapeutic antitumor vaccine, or exvivo to stimulate a population of T cells, NK cells or NKT cells foradoptive therapy of cancer (Examples 29).

42. DCs Expressing SAg and Tumor Associated Antigens—Production byProcessing of Apoptotic Tumor Cells or Tumor Cell Lysates

DCs expressing SAg and tumor associated antigens are prepared withoutcell fusion (Example 28). Apoptotic, SAg transfected tumor cells areprepared by first transfecting tumor cells with SAg (Example 1) and theninducing apoptosis by irradiation or other methods well known in the art(Example 28). DCs express αv□₅-binding integrins and secretethrombospondin which ligates vitronectin expressed on the surface of theapoptotic tumor cell. DC surface CD36 binds to its natural ligand,sequestrin, also expressed on apoptotic tumor cells. The apoptoticSAg-expressing tumor cells are phagocytosed and processed by DCs underconditions described in Example 28.

In another embodiment, lysates of tumor cells optionally expressing SAgare also used as above. Tumor cells are first transformed to express SAgand then lysed (Example 28. These lysates are “fed” top DCs as inExample 28. DCs treated in this way can now present tumor associatedantigens along with SAg to the immune system. Alternatively, DCs arefirst transformed to express SAg, and these cells are allowed tophagocytose or process apoptotic tumor cells or lysates. Optionally thetumor cells may have been previously genetically modified with nucleicacids so that they synthesize □1,3-glucan, LPS, peptidoglycan or GalCer. The resulting SAg-expressing DC, after phagocytosing apoptotictumor cells or lysates, expresses MHC class II, costimulatory moleculesCD40, CD80 and CD86, together with SAg and tumor associated antigen. Theadditional expression of SAg in this system permits more potentactivation of T cells, NKT cells and NK cells which recognize the tumorassociated antigens expressed on the DC surface in the context of MHCand costimulatory molecules. Such DCs are used in a preventative ortherapeutic antitumor vaccine (Example 29) or ex vivo to activate a Tcells, NKT cells or NK cells for the adoptive immunotherapy (Example29).

43. DCs Expressing or Secreting SAg Cotransfected with a TumorAssociated Antigen or “String of Beads” Tumor Antigens

When a dominant tumor associated antigen (protein) is known, nucleicacid encoding such an antigen are used to transform DCs which alreadyexpress or secrete SAg (Example 35). Antigens identified by “SELEX”technology which consists of nucleic acids encoding tumor antigens fromdistinct structural and functional categories of human tumor associatedantigens, including mutants, differentiation variants, splice variants,amplified/overexpressed antigens or retroviral antigens may be used.Nucleic acids encoding tumor antigens used to transfect SAg-expressingDCs or DC/tc hybrids. This invention contemplates transfecting withindividual nucleic acids encoding a single antigen, or multiples as in a“string of beads” carried by adenoviral or other vectors known in theart (Example 35). Nucleic acids encoding a “string of beads” or tumorassociated antigens identified by SERAX may be fused in frame (orcotransfected with) SAg-encoding nucleic acid into DCs or DC/tc. TheseSAg- and tumor antigen-expressing DCs or DC/tc hybrids are used as apreventative or therapeutic antitumor vaccines (Example 29) or asstimulators ex vivo of T cells, NKT cells or NK cells for adoptiveimmunotherapy (Example 29).

Furthermore, nucleic acids encoding proteins listed in Tables I, II, IVand V, for example, angiostatin, protein A, erb/Neu and HSPs,staphylococcal collagen adhesin, are introduced into and expressed intumor cells or DCs that express or secrete SAg, or into S/D/t cells.These cells that coexpressing the proteins and peptides of Tables I, II,IV and V together with SAg are useful as preventative or therapeuticantitumor vaccines (Example 29) or as stimulators ex vivo that activateT cells, NKT cells or NK cells for adoptive immunotherapy (Example 29).

44. Naked DNA or RNA Obtained from the Various Cells Described Abovethat Express and/or Secrete SAg

DNA containing the CpG backbone is extracted from tumor cells or DCsthat express/secrete SAgs or S/D/t cells (Example 30-34). The preferredsource of DNA or RNA is the S/D/t cells DCs or tumor cells expressingSAg are also useful. Alternatively, the DNA or RNA can be obtained fromDCs, tumor cells or DC/tc into which SAgs were introduced by the cellshaving phagocytosed SAg-transformed apoptotic tumor cells or tumor celllysates.

The extracted DNA or RNA is used as a naked DNA or RNA preventative ortherapeutic vaccine (Examples 30-34). Alternatively, this nucleic acidmaterial may be used ex vivo to activate T cell, NKT cells or NK cellsadoptive immunotherapy (Example 1, 31, 33). This extracted DNA or RNAmay be used in an initial step of inducing immune reactivity in regionallymph nodes of tumor bearing subjects. After this “priming,” T cells,NKT cells and/or NK cells are harvested from these lymph nodes, expandedin culture in the presence of additional SAg, SAg-expressing DC or tumorcells, or S/D/t cells to generate a T cell, NKT cell or NK cellpopulation for adoptive immunotherapy (Examples 29). DNA or RNA forimmunization may also be obtained from the various cells described abovethat express SAg, and which additionally express or severalStaphylococcal adhesins, □-glucans, LPS, peptidoglycans, teichoic acids,mannose, mannan, protein A and/or their respective binding proteins.

Also useful for naked nucleic acid immunization are bacterial or insectnucleic acids (with CpG motifs) which encode enzymes that catalyze thebiosynthesis of □-1,3-glucans, LPS, peptidoglycan, -Gal, GalCer,teichoic acids, mannan or mannose. Also useful are bacterial or insectnucleic acids that encode the binding proteins for the abovecarbohydrate-based molecules, glycoprotein lectins that bind thecarbohydrate structures, or protein A. Such nucleic acids are used toco-immunize along with SAg expressing DCs or tumor cells or S/D/t cells.Such combined vaccine preparations are used as a preventative ortherapeutic antitumor vaccines (Examples 29, 30). Alternatively, theymay be used to initiate adoptive T cell therapy by priming regionallymph nodes T cells which are harvested, expanded in vitro bystimulation with S/D/t cells, accompanied by, or followed with IL-2. Thetumor antigen-sensitized T cells are reinfused into subjects asdescribed in Example 29.

45. Exosomes Derived from (1) SAg-Expressing Tumor Cells (2) SAgExpressing-DCs (3) S/D/t cells or (4) DC/tc Hybrid Cells

MHC-peptide complexes accumulate in endosomes and lysosomes, whichcompartments contain MHC class II-enriched internal vesicles that arereleased outside the cell following direct fusion of the externalendosomal membrane with the plasma membrane. These vesicles, termed“exosomes” are capable of stimulating CD4+ T cell clones in vitro. Inaddition, tumor peptide-pulsed DC-derived exosomes prime specific CTLsin vivo leading to a T cell-dependent eradication or suppressed growthof established murine tumors. In the present invention, the exosomeswhich have SAgs in addition to tumor associated antigens and MHC class Iand class II molecules are prepared. Such preparations are significantlymore potent in their ability to induce shrinkage of established tumorsand prevent tumor outgrowth.

Exosomes are prepared from (1) tumor cells or DCs which have beentransfected with SAgs (2) S/D/t cells, (3) DCs or hybrid DC/tc whichhave phagocytosed SAg-expressing apoptotic tumor cells or tumor celllysates (Example 36). In the above hybrids, either the DC or tumor cellmay be transfected with SAg-encoding nucleic acid prior to fusion. Theresulting exosomes express MHC class I and class II molecules, SAgs andtumor associated antigen. In order to ensure the routing ofthetransforming SAg to exosomes, the SAg-encoding nucleic acid shouldinclude a sorting signal to localize the SAg to the exosome. These cellsmay be pulsed with tumor associated antigens shortly before isolation oftheir exosomes. The isolated exosomes are used as preventative ortherapeutic antitumor vaccines (Example 36) or as stimulators ex vivothat activate T cells, NKT cells or NK cells for adoptive immunotherapy(Example 36). These various exosome preparations are extremely effectiveinducers of anti-tumor responses.

46. Cell surface Display of Recombinant SAg and Tumor AssociatedAntigens in Bacteria

Heterologous proteins and various carbohydrate-containing moieties,displayed on the surface of bacterial cells often act as major antigenicsystems that stimulate anti-tumor immunity. Such antigens includeGalCer, αGal, □1,3-glucans, LPS, peptidoglycans, teichoic acids andmannan. These structures will be referred to below collectively as“anti-tumor motifs.” These structures are created by the action ofenzymes encoded by a number of bacterial and fungal genes. For example,Sphingomonas paucimobilis expresses GalCer, or Klebsiella aerobacterexpresses -Gal and LPS, and Cryptococcus expresses □1,3-glucan. Becausenot all the genes responsible for the biosynthesis of these moleculeshave not been identified, it is difficult to isolate them and introducethem into mammalian cells. These structures are, however, biosynthesizedin abundance by bacteria. Immunization with live recombinant bacteriainduces both local and systemic immune responses suggesting thatgram-positive bacteria might constitute potential live bacterial vaccinedelivery systems. The surface molecules of gram-positive bacteria seemto be more permissive for the insertion of extended sequences of foreignproteins than are gram-negative bacteria, in which both translocationthrough the cytoplasmic membrane and correct integration into the outermembrane are required for proper surface exposure.

In the present invention, different bacterial surface display systemsare used to express natural anti-tumor motifs for developing livebacterial vaccine vehicles. SAgs are provided to bacteria which do notnaturally bio synthesize them so that they are expressed together withnatural anti-tumor motifs made in the bacteria. These bacteria are thenused as preventive or therapeutic antitumor vaccines (Example 28).

Sphingomonas paucimobilis bacteria express GalCer which can activate theV14 invariant chain expressed by NKT cells. These cells recognizes thegalactosylceramide epitope. NK cells, using their NKP1-1 receptors,recognize carbohydrate units such as 1□,3-glucans expressed widely onfungi. NK cells are activated directly by SAgs. Further proliferation isinduced by interferon produced by T cells in response to the SAg. Humanshave natural antibodies specific for the αGal epitope. This epitope isconstitutively expressed on several bacteria including Klebsiellaaerobacter and E. coli.

Coexpression of SAg with the above anti-tumor motifs in recombinantbacteria or fungi provides potent signals to activate NKT cells, T cellsand NK cells and to induce production of TH-1 cytokines. The adhesionmolecule VCAM-1 expressed by some SAgs such as enterotoxin C contributesto the process by costimulation. Therefore, the SAg expressing bacteria(whether natural or transformed) are capable of activating all of themajor cell types involved in the anti-tumor response.

In the present approach, the preferred SAg is SEB. SEB is introduced forsurface display into S. carnosus. E. coli-staphylococcus shuttle vectorsare constructed by taking advantage of (1) the promoter signal sequenceand propeptide region from the lipase gene construct derived from S.hyicus and (2) the cell surface attachment part of staphylococcalprotein A. A 198-amino-acid region, designated ABP (albumin bindingprotein), is expressed adjacent to the cell wall to increaseaccessibility to the surface-displayed target peptides. Staphylococcalenterotoxin B is introduced between the lipase propeptide and the ABPregion and the surface exposure of the three different regions aretested separately with different assays.

These recombinant bacteria are useful as a preventative or therapeuticantitumor vaccine (Example 28) or as stimulators ex vivo that activate Tcells, NKT cells or NK cells for adoptive immunotherapy (Example 28).

47. Introduction of Staphylococcal Collagen Binding Adhesins into DCs,Tumor Cells or S/D/t Cells

Nucleic acids encoding SAgs are transfected into these various cells, asdescribed above, together with nucleic acids encoding Staphylococcalcollagen adhesin(SEQ ID NOS:44-45). Mice immunized with a recombinantfragment of the collagen adhesin were protected against Staphylococcusaureus-mediated septic death. Sera from S. aureus-immunized micepromoted phagocytic uptake (opsonized) and enhanced intracellularkilling of the bacteria compared to sera from control mice.

The collagen binding adhesin is isolated from S. aureus strain Cowan.Sequencing of the cloned corresponding gene cna revealed a 133-kDapolypeptide (close to that of 135 kDa reported for the native protein).This protein is proposed to consist of a signal sequence (S) followed bya large nonrepetitive region (A). Immediately following the A region arethree consecutive repeats of a 167 amino acid long unit (B1, B2, B3). Acell wall (W) region consisting of 64 amino acid proline-and lysine-richdomain is followed by stretch of hydrophobic amino acids (M), presumablyconstituting the cell membrane spanning region. Finally, the C-terminus(C) is made up of a few positively charged amino acids. This modelstructure is used as the starting point to identify the collagen bindingdomain. The ligand binding site is localized within the 135-kDa S.aureus collagen adhesin. The collagen binding domain is localized to a168 amino acid long segment [CBD (151-318)] within the N-terminalportion of the adhesin. Using biospecific interaction analysis, bovinecollagen was found to contain eight binding sites for CBD (151-318), twoof which were high affinity and six low affinity. The deduced amino acidsequence of the ligand binding domain of the collagen adhesin ispresented. Subsequently a discrete collagen-binding domain within thecollagen adhesin was identified and localized to a region between aminoacids Asp209 and Tyr233. The FDA strain 574 of S. aureus encodes a 1185amino acid collagen adhesin. The complete nucleotide sequence of the cnagene as well as a schematic model of the collagen adhesin have beenpublished. The overall structure resembles that of other gram positivesurface structures. The lysine and proline rich hydrophilic region whichfollows the repeated domains resembles a structure in protein A,staphylococcal fibronectin receptor and streptococcal protein G and Mproteins. Also present is the hexapeptide LPKTGM which is similar to theconsensus sequence LPXTGE which is conserved among other gram positivesurface proteins. The hydrophilic region is thought to mediate thebinding of the protein to the cell wall. The presence of hydrophobicamino acids which may traverse the membrane followed by a C-terminalcluster of positively charged residues, possibly located on thecytoplasmic side of the membrane, is characteristic of staphylococcalcell surface proteins. In the collagen adhesin, a 29 amino acid signalpeptide at the N-terminus is followed by a large nonrepetitive A domain,and the highly homologous domains B1, B2 and B3 (probably a result of aseries of stepwise gene duplication events). Collagen binding receptorshave been found on other species of bacteria such as the 75X adhesin ofuropathogenic E. coli. Type 3 fimbrias from pathogenic enteric bacteria,some species of oral streptococci Streptococcus pyogenes, Yersinia andTreponema pallidium have all been reported to bind various forms ofcollagen. Thus the collagen binding appears to be a common modality usedby pathogenic bacteria of a diverse group to adhere selectively to hosttissues and form a focus of infection.

Nucleic acids encoding staphylococcus collagen adhesin are introducedinto SAg-expressing tumor cells or DCs, or S/D/t cells. The cellsco-expressing the staphylococcal collagen adhesin with SAgs are usefulas a preventative or therapeutic antitumor vaccines (Example 28) or asstimulators ex vivo that activate T cells, NKT cells or NK cells foradoptive immunotherapy (Example 28).

48. Co-Expression of Anti-Tumor Motifs or their Binding Proteins withSAg

Tumor cells or DCs expressing SAgs, or S/D/t cells, are transformed withnucleic acids encoding enzymes that catalyze the biosynthesis ofanti-tumor motifs, including the αGal epitope, the GalCer epitope,□-1,3-glucans, LPS, peptidoglycan, teichoic acids or a protein orpeptide such as Staphylococcal adhesins, protein A, and/or the bindingproteins for the above motifs or proteins. Transformation may be achieveusing bacterial plasmids or nucleic acids integrated into an appropriateviral vector. These antigenic structures are fundamental unitsrecognized in the primitive host defense mechanisms (“innate immunity”)of invertebrates, but also evoke responses in mammalian immune systemsvia the TOLL and NFκB systems.

DNA encoding the galactosyltransferase that synthesizes the saccharidestructure containing the αGal epitope, and gene clusters encoding thebiosynthetic pathway for LPS are described in Schnaitman C A, et al.,Microbiol. Rev. 57: 655-682 (1993). DNA is extracted from bacteria whichbiosynthesize these molecules and used to transfect DCs, tumor cells, orS/D/t cells For creation of the GalCer structure, the source of DNA isSphingomonas paucimobilis organisms. Nucleic acids encoding the pathwaysfor biosynthesis of □-1,3-glucans, peptidoglycans, and protein A havebeen cloned from insects and Staphylococcus aureus, respectively. Thesenucleic acids are cloned into suitable expression vectors and introducedinto the target cells. Resulting S/D/t cells thus express SAg as well asthe anti-tumor motif structure.

S/D/t cells that co-express Gal can interact with and stimulate NKTcells through the Vα14 invariant chain which naturally recognizes the-galactosylceramide epitope. NK cells, via their NKP1-1 receptors, willrecognize carbohydrate units such as □-1,3-glucans on the S/D/t cells.The co-expressed SAg induces further NKT cell expansion. The SAg is alsocapable of inducing massive proliferation of conventional T cells whichcan be further promoted by the co-expression of B7-1, B7-2 and ICAM-1which are normally expressed on DCs. VCAM-1, expressed by some SAgs suchas enterotoxin C, also is capable of contributing to this stimulation.As indicated above, NK cells are activated directly or indirectly byT-cell derived interferon.

The S/D/t cells (as well as tumor cells or DCs expressing SAg) that alsoexpress one or more of the anti-tumor motifs are capable of activatingall of the major cell types involved in anti-tumor immunity: T cellsspecific for peptides, NKT cells reactive with lipoproteins andglycosylceramides and NK cells that recognize for oligosaccharides.These cells are useful as preventative or therapeutic antitumor vaccines(Examples 29) or as stimulators ex. vivo that activate T cells, NKTcells or NK cells for adoptive immunotherapy (Example 29).

49. Sags Combined with Low Density Lipoproteins (LDL), Oxidized LDL (oxyLDL) Oxidized LDL Mimics and Apolipoproteins

In the present invention, low density lipoproteins (collectively LDL)intermediate density LDL (IDL), chylomicrons, very low densitylipoproteins (VLDL), oxidized LDL (oxyLDL), oxyLDL mimics as well as andapolipoproteins including but not limited to apolipoprotein (a), B100and E4 are conjugated to superantigens and are useful as anti-canceragents alone.

LDLs, oxyLDLs and apolipoproteins are physically trapped or bind toreceptors expressed in the dense network of randomly branching bloodvessels and sinusoids of the tumor neovasculature and have the capacityto deposit or bind to LDL receptors on the tumor endothelium and toscavenger recepors on macrophages OxyLDL or apolipoproteins bound totumor endotheium or macrophages they induce apoptosis or they promoteinflammation by activating vascular cells and macrophages to generatecytokines, chemoattractants and tissue factor.

Superantigens in nucleic acid or polypeptide form are conjugated to thelipoproteins and amplify the inflammatory effect of the lipoproteins byinducing apoptosis of endothelial cells, upregulating endothelial cellintegrins, adhesins and procoagulant activity whicle activatingmacrophages and immunocytes. Any tumor which is neovascularized iseligible for this therapy. These conjugates therefore have theadvantages of localizing to disseminated and neovascularized tumor,inducing apoptosis and initiating a powerful anti-tumor response.

Lipoproteins

Lipoproteins are globular particles of high molecular weight thattransport nonpolar lipids (primarily triglycerides and cholesterolesters) through the plasma. Lipoproteins have been classified on thebasis of their densities into five major classes: chylomicrons, very lowdensity lipoproteins (VLDL), intermediate-density lipoproteins (IDL),low-density lipoproteins (LDL), and high-density lipoproteins (HDL). Thephysical-chemical characteristics of the major lipoprotein classes arepresented in Table The core of the spherical lipoprotein particle iscomposed of two nonpolar lipids hydrophobic lipids, triglyceride andcholesteryl ester, which are present in different lipoproteins invarying amounts. This hydrophobic core accounts for most of the mass ofthe particle, and consists of triglycerides and cholesterol esters invarying proportions. Surrounding the core is a polar surface coat ofphospholipids that stabilize the lipoprotein particle so that it canremain in solution in the plasma. Variable amounts of unesterifiedcholesterol are interdigitated with the phospholipids of the surfacecoat. In addition to phospholipid, the polar coat contains small amountsof unesterified cholesterol. Each lipoprotein particle also containsspecific proteins (termed apoproteins) that are exposed at the surfaceand extend into the core. The apoproteins bind specific enzymes orreceptors on tumor microvascular cells.

Chylomicrons

Chylomicrons are large lipoprotein particles formed within intestinalepithelial cells from dietary triglycerides and cholesterol which aresecreted into the intestinal lymph and pass into the general circulationwhere they adhere to LDL receptors on the tumor microcapillaries.Chylomicron remnants are removed by both LDL receptors and LDL- receptorrelated protein/alpha2-macroglobulin receptor (LRP). While bound tothese endothelial surfaces, the chylomicrons are exposed to the enzymelipoprotein lipase. The chylomicrons contain an apoprotein, apoproteinCII, that activates the lipase, liberating free fatty acids andmonoglycerides.

Very Low Density Lipoprotein (VLDL)

Very low density lipoprotein (VLDL)are triglyceride rich particles whichare secreted from the liver into the bloodstream after conversion ofcarbohydrate to glycerol-esterified fatty acids to form triglycerides.VLDL particles are relatively large, carry 5 to 10 times moretriglycerides than cholesteryl esters, and contain a form of apoproteinB, designated B 100, that differs from the apoprotein B48 ofchylomicrons. The VLDL particles are transported to LDL receptors ontumor microcapillaries, where they interact with the same lipoproteinlipase enzyme that catabolizes chylomicrons. VLDL also binds to the VLDLreceptor via apolipoprotein E and lipoprotein lipase. Bothapolipoprotein E and lipoprotein lipase are constituents of chylomicronremnants which are a physiological ligand for the VLDL receptor.

Plasma Apolipoproteins

Plasma apolipoproteins have a central role in plasma lipid transport.Central to the functions of all apolipoproteins (apo) is specializedregions termed amphiphathic helices which have the ability to bindphospholipids. The amphiphatic helices in apoA-I, apoA-II, and apoC-IIIcomprise multiple repeats of 22 amino acids or 22-mer periodicity eachconsisting of a tandem array of two 11-mers which tend to begin or endwith a proline. The characteristic spatial arrangement of thehydrophobic and hydrophilic amino acids within the amphipathic helicesis that the hydrophobic face is intercalated between the fatty acylchains of phospholipids and the hydrophilic face is located close to thepolar head groups of phospholipids. Such an orientation allows theinteraction of protein domains with lipoprotein-modifying enzymes andcellular receptors that control the catabolism of lipoproteins (Lp) andtheir removal from the circulation. The major apolipoproteins useful inthe present superantigen-apolipoprotein conjugates are as follows:

Apolipoprotein (a)

Apolipoprotein (a) (Lp (a)) is made by hepatocytes and is secreted intoplasma where it forms a covalent linkage by a single interchaindisulfide bond to a unique multikringle glycoprotein, with apo B 100 ofLDL to form lipoprotein(a). called aploliprotein(a). Protein apo (a) hasstructural similarities to plasminogen and consists of multiple bentrepeats of amino acid sequences. Apolipoprotein (a) exists in polymorphsdistinguished by molecular weights. The molecular basis for the sizevariation of apo[a] is primarily due to multiple apo[a] alleles thatdiffer in the number of kringle type 2 (plasminogen kringle type 4)repeats. Minor variability in apo(a) size might be due to differences inglycosylation, as carbohydrates make up 25-40% of the apo (a) weight.

A close structural similarity exists between apo(a) and plasminogen aprotease zymogen whose active form cleaves fibrin to dissolve bloodclots, is activated by tissue and urokinase plasminogen activators viacleavage at a specific arginine residue. Indeed, in-vitro and ex-vivostudies have shown that apo(a) binds to immobilized fibrin (fibrinogen),to the plasminogen receptor on endothelial cells and competes withtissue plasminogen activator in converting plasminogen to plasmin.Lipoprotein(a) also competes with plasminogen for its high-affmitybinding sites in endothelium, platelets, and macrophages. Because ofstructural homology with plasminogen apo(a)I competively inhibitsfibrin-dependent activation of plasminogen to plasmin andplasmin-mediated activation of cytokine transforming growth factor-□.Hence, Lp(a) is capable of interfering with the fibrinolytic process byacting as a procoagulant. The colocalization of apo(a) with fibrin(fibrinogen) in the arterial wall further suggests that Lp(a) isthrombogenic.

Lp(a) is a poor ligand for the LDL receptor and is consequently taken upand degraded by unregulated mechanisms, leading to tissue accumulation.Lp(a) is targeted to uptake by macrophages, presumably through thescavenger-receptor pathway. Owing to the lower B-carotene content, Lp(a)may be more easily oxidized than LDL. Oxidized Lp(a) such as Lp(a)modified by malondialdehyde, a product generated in vivo from aggregatedplatelets, is avidly taken up by monocyte-macrophages. through thescavenger-receptor pathway. Lp(a) accumulates in either the arterialwall and in vein grafts, respectively suggesting that Lp(a) can alsotraverse the endothelium of arterial vessels and reach the intima bynon-receptor-mediated mechanisms and that this transport process isinfluenced by the density/size of Lp(a). There, Lp(a) can form complexeswith such tissue-matrix components as proteoglycans, glycosaminoglycans,and collagen as well as fibrin. The magnitude of the transfer of Lp(a)from the plasma compartment to the arterial wall is larger when plasmaLp(a) levels are elevated because of a gradient effect or because of apossible direct action of Lp(a) on arterial permeability.

Apolipoprotein B

Apolipoprotein B occurs in two forms termed apoB-100 and apoB-48. Inhumans apoB-48 is produced only by the intestine and apolipoproteinB-100 originates from the liver. Apolipoprotein B-100, which contains4536 amino acid residues, is the major apolipoprotein of VLDL, IDL,Lp(a) and is the sole apolipoprotein of LDL. ApoB-48 consists of theamino-terminal half of apoB-100, contains 2152 amino acid residues andis devoid of binding domain for the LDL receptor.

Apolipoprotein E4

Apolipoprotein (apo) E is a 34-kCa protein coded for by a gene onchromosome 19 and plays a prominent role in the transport and metabolismof plasma cholesterol and triglyceride through its ablity to interactwith the low density lipoprotein (LDL) receptor and the LDL receptorrelated protein (LRP). Apolipoprotein E (apoE) is a 34-kda proteincomponent of lipoproteins that mediates their binding to the low densitylipoprotein (LDL) receptor and to the LDL receptor-related protein(LRP). Apolipoprotein E is a major apolipoprotein in the nervous system,where it is thought to redistribute lipoprotein cholesterol among theneurons and their supporting cells and to maintain cholesterolhomeostasis. Apart from this function, apoE in the peripheral nervoussystem functions in the redistribution of lipids during regeneration.

Oxidized LDL

LDL is also rapidly transported across an intact endothelium and becomestrapped in the three-dimensional cage work of fibers and fibrilssecreted by the artery wall cells. This concentration-dependent processdoes not require receptor-mediated endocytosis. LDL entrapped inarteries or bound to receptors on endothelium or the tumormicrocirculation undergoes diverse enzymic and chemical modifications.It can also be introduced into the cell a variety of lipophilic invaderssuch as lipid peroxidation products and cholesterol oxides that mayirreversibly modify cellular functions. The early oxidative modificationof the trapped LDL in vivo occurs before monocytes are recruited andresults in the oxidization of lipids in LDL with little change in apoB.

Monocytes recruited to the lesion, are converted into macrophages andthe LDL lipids are further oxidized. Once the LDL contains fatty acidlipid peroxides, there follows (especially in the presence of metalions) a rapid propagation that amplifies dramatically the number of freeradicals and leads to extensive fragmentation of the fatty acid chainswith the generation of a broad spectrum of oxysterols, shorter-chainaldehydes (e.g., malondialdehyde and 4-hydroxynonenal) some of whichinvolve the covalent binding of short-chain substituents to the aminogroups of lysine residues in apoprotein B (and possibly to otherportions of the apoprotein B molecule) masking lysine 6-amino groups.Acetyl LDL and scavenger receptors recognize modifications effected bychemical acetylation and highly oxidized LDL.

Incubation of LDL with endothelial cells, smooth muscle cells, andmacrophages in vitro induces oxidation of polyunsaturated fatty acids.Lipid peroxides formed fragment fatty acyl chains and attach covalentlyto apoB or fragments thereof, thereby rendering the modified particlescompetent for endocytosis by the scavenger receptor. LDL particles alsoundergo peroxidation of polyunsaturated fatty acids which producesoxidative modification and conversion of LDL lecithin to lysolecithin.

Modification of LDL with malondialdehyde, a product of arachidonic acidmetabolism or oxidation of LDL leads to foam cell formation. Unlikenative LDL, oxidized LDL is mitogenic or induces apoptosis in arterialendothelial and smooth muscle cells. It also induces endothelial cellsand monocytes to express high levels of tissue factor and plasminogenactivator inhibitor. Levels of P-selectin are increased intracellularlyand are released by oxy-LDL which can also directly stimulate PDGFproduction in endothelial cells. Oxidized LDL also induce the expressionof endothelin, to inhibit the expression of nitric oxide synthase, andto inhibit the resulting vasodilation. Platelet accumulation and localincreases in thromboxane A, serotonin, ADP, platelet activating factor,and activated thrombin, together with a local reduction in prostacyclinfurther contribute to a procoagulant state.

Another stable end product of cellular oxidative modification of LDL islysophosphatidylcholine, which is generated by phospholipase A2hydrolysis. This lipid selectively induces the expression of adhesionmolecules for monocytes, vascular cell adhesion molecule-1 (VCAM-1), andICAM-1 in cultured human arterial endothelial cells. TNF-a activation isa prerequisite for the observed lysophosphatidylcholine induction ofVCAM-1. Lysophosphatidylcholine also induces monocyte chemotaxis,arrests macrophage migration and induces macrophage proliferationthrough SR-A-mediated internalization of modified lipoprotein. Finally,lysophosphatidylcholine induces gene expression for smoothmuscle/fibroblast growth factors, the A and B chains of PDGF, andheparin-binding epidermal growth factor-like protein in culturedendothelial cells.

oxy LDL Mimics

The cytotoxic effects of highly oxidized LDL are mimicked by higherconcentrations of oxysteroid. particularly 7b-hydroperoxycholesterol.7b-hydroxycholesterol, 7-ketocholesterol and 5a-6a-epoxycholesterol.These oxysterols can induce apoptosis in a variety of cells. Of theseend products, 73-hydroperoxy-choles-5-en-3B-ol has been identified asthe primary cytotoxin in highly oxidized LDL. This molecule accounts forapproximately 90% of the cytotoxicicy of lipids extracted from highlyoxidized LDL in vitro. Fatty acid hydroperoxides and aldehydes found inoxidized LDL also alter intracellular functions. For example,4-hydroxynonenal (4-HNE). a component of oxidized LDL, induces bindingof the coagulation protein, Factor Xa to endothelial cells. In addition,oxidized LDL and mm-LDL can significantly induce the release of IL-1from macrophages. Saponified Cu²+-oxidized LDL and mm-LDL have beenshown to contain 9-HODE, 13-HODE, and cholesterol-9-HODE, which increaseIL-1 release from macrophages. 4-HNE also causes a variety of effects onmonocytes, including stimulation of monocyte migration through inductionof chemoattractant proteins and initiation of apoptosis

Mildly Oxidized LDL (mm-LDL)

Mildly oxidized LDL (mm-LDL) induces elevated levels of cAMP by a Gprotein-mediated mechanism and induces inflammatory molecules both byincreasing the rates of gene transcription and by stabilizing the MRNAfor these genes. Exposure of the arterial wall to (mm-LDL) orbiologically active products of lipid peroxidation results in binding tothe LDL-R. mm-LDL also induces monocytes to bind to endothelial cells.and induces changes which affect monocyte binding, tethering,activation, and attachment. mm-LDL also induces an inflammatoryphenotype in endothelial cells and proinflammatory cytokines accompaniedby increase the levels of the transcription factor, NF kB, which hasbeen linked to the expression of a variety of adhesion molecules. Inparticular, lysophosphosphatidylcholine, a product of LDL oxidation, hasbeen shown to be a chemoattractant for monocytes and T-lymphocytes, toinduce the adhesion molecules VCAM-1 and ICAM-1, and to increase levelsof PDGF and heparin-binding epidermal growth factor mRNA in endothelialcells and smooth muscle cells. Increases in ICAM-1 expression lead toenhanced monocyte adhesion to the vessel wall.

Moreover, mm-LDL induces endothelial cells to produce the potentmonocyte activators monocyte chemoattractant protein 1 (MCP-1) andmonocyte colony stimulating factor (M-CSF). Macrophage Class A scavengerreceptors and CD36, a Class B scavenger receptor are up-regulated byM-CSF. Once bound to specific scavenger receptors, mm-LDL can initiatecell signaling events in vascular cells stimulating phosphoinositidemetabolism and calcium flux as well as stimulate phospholipase E1activity through a tyrosine kinase-dependent mechanism independent ofprotein kinase C. This induces the release of phosphatidic acid orarachidonic acid for eicosanoid production in the vessel wall. A portionof this activity may be mediated by the Class A scavenger receptorligands which stimulate macrophage urokinase expression and IL-1production a growth factor for smooth muscle cells.

The biological properties of the lipids in mildly oxidized LDL differfrom those induced by the lipids in highly oxidized LDL. For example,the expression of tissue factor by endothelial cells is induced bymildly oxidized LDL but not by highly oxidized LDL. The lipids in highlyoxidized LDL are cytotoxic, whereas the lipids in mildly oxidized LDLare not. Mildly oxidized LDL induced the activation of the NFKB-liketranscription factor and the increase in the appearance of specificoxidized phospholipids. With continued oxidation, highly oxidized LDLsuch as lysophosphatidylcholine and oxidized sterols are produced withdifferent biological activity as given above.

The ability of mm-LDL to induce monocyte adherence to endothelial cellsis mimicked by three polar bioactive lipids isolated from mm-LDL as wellas oxidized 1-palmitoyl-2-arachidonyl-sn-glycerophosphocholine. Themolecular structure of two bioactive lipids were identified1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphocholine (m/t 594.3) and1-pahnitoyl-2-glutaryl-sn-glycero-3-phosphocholine (m/t 610.2). Thethird lipid (m/t 831) has tentatively been described as an arachidonicacid-containing phospholipid containing three or four oxygen molecules,potentially forming a conjugated triene structure characteristic ofleukotrienes. The latter serves as a substrate for paraoxonase, andthose with fragmentation products such as 5-oxyvalerate at the sn-2position may represent substrates for PAF acetylhydroxylase.

Glycated LDL

Glycated LDL is recognized less well by the LDL receptor, but is takenup more rapidly by macrophages. Very prolonged exposure of LDL to highconcentrations of glucose leads to glucose-mediated cross-linking andthe generation of advanced glycosylation end products, which macrophagesrecognize in a specific saturable fashion.

Artificial Complexes of LDL

Artificial complexes of LDL formed by incubation with fibronectin,heparin, and fibrillar collagen are also candidates, and the uptakethere appears to be through recognition of the fibronectin. Complexes ofLDL with itself are taken up more rapidly than native LDL via the LDLreceptor. After incubation with neutrophils LDL is taken up more rapidlyby macrophages. This is attributable to the dimerization of LDL by theaction of secreted neutrophil elastase on native LDL

Apoprotein Genes

The genes for the major apoproteins associated with the lipoproteinshave been cloned. These include apolipoprotein (a) (McLean J W Nature330: 132-137 (1987)), apolipoprotein B-100 (Chen S H J. Biol Chem. 261:12918-12921 (1986)), apolipoprotein E4 provided by Drs. S Lauer and J.Taylor. Lp(a) has been cloned from cDNA libraries constructed from humanliver mRNA (McLean J W Nature 330: 132-137 (1987)). Complete sequenceanalysis of a 14 000-base-pair (bp) DNA copy of apo(a) mRNA showed manyexact or nearly exact repeats of a 342-bp sequence occurred. Indeed,most of the mRNA consist of 22 tandem exact repeats and 15 modifiedrepeats. Apolipoprotein (a) belongs to a gene family that includes genesencoding clotting factors, structural proteins, and growth factors.Domains shared by these proteins are protease-like domains, kringleunits, calcium binding domains, and epidermal growth factor precursordomains.

In the present invention, superantigens are ligated to the major classesof lipoproteins in human plasma including LDL, IDL, HDL, VLDL,chylomicons and remnants containing apoproteins and mm-LDL, oxy LDLisosterols, inositols, lysophosphatidlycholine, synthetic mimics of LDLactivity and oxyLDL byproducts by methods given in Example 47 Because oftheir unique capacity to adhere to tumor niicrovasculature and evoke anapoptotic/inflammatory/prothrombotic response, the lipoproteinstructures preferred for ligation to SAg include but are not limited toLp(a), LpB-1000 or B-47, oxyLDL, oxyLDL byproducts, oxyLDL mimics andIDL.

The lipoproteins used for conjugation are prepared as in Examples 48-49.The superantigens used for conjugation are preferentially in nucleicacid or phage form but may also be in peptide, polypeptide nucleic acidor phage display form. They are coupled to the various LDL, oxyLDL orapolipoproteins via methods given in Examples 3, 5, 47. Alternatively,SAg are incorporated or bound or conjugated to a vesicular, exosomalstructures shed from normal, tumor or sickled cells expressing LDL, oxyLDL, oxyLDL mimics or apolioproteins. Superantigens are also integratedinto liposomal structures prepared to express natural or synthetic LDL,oxyLDL, apolipoprotens or oxyLDL mimics as described in Section 45 andExamples 3, 5, 6, 36, 42. Optionally, integrin ligand sequences such asRGD are added to facilitate the localization of the conjugates to thetumor microvasculature binding to the a_(v)b₃ integrin and a_(v)b₅integrin which are expressed therein (see Example 6). Thesesuperantigen-lipoprotein conjugates are physically trapped in the densenetwork of randomly branching blood vessels of the tumormicrocirculation and also bind to LDL or scavenger receptors expressedin the tumor neovasculature.

Constructs consisting of naked Sag nucleic acids containing CpG backbonefused to apoprotein nucleic acids alone or incorporated into liposomesare prepared as in Example 3, 6, 14, 30-31 and delivered to the tumorsites in vivo as in Examples 14, 30-31.

These constructs are useful in vivo as a therapeutic antitumor vaccinesaccording to Examples 14, 15, 16, 18-23. They are also useful ex vivofor producing a population of tumor specific effector T or NKT cells foradoptive immunotherapy of cancer (Examples 2-5, 7, 15, 16, 18-23).

Tumor Cells or Sickled Erythrocytes and Vesicles Expressing SAg andApolipoproteins

Superantigen nucleic acids are fused in frame to nucleic acids encodingapoproteins including but not limited to apoproteins Lp(a), B-48 and 100and E3 and transfected into tumor cells in vivo to produce tumor cellsexpressing superantigens and apoproteins. These tumor cells arerecognized by apoprotein receptors in tumor microvasculature. Tumorcells are also transfected ex vivo with the identical nucleic acidconstructs. A RGD sequence is added to promote deposition in the tumormicrovasculature which are useful. These tumor cell transfectantsexpressing Sag, apoprotein and RGD bind to apoprotein receptors andintegrins respectively expressed in tumor microvasculature wherein theyinitiate a potent and localized anti-tumor response.

Superantigen nucleic acids together with nucleic acids encoding eitherapo(a), apoB and apoE4 are also transfected into nucleated sicklederythrocytes (e.g., proerythroblast or normoblast phase) by methodsgiven in Examples 1 and 6. The integrin ligand RGD nucleic acids aretransfected into tumor cells or sickled cells to facilitate thelocalization of the transfected tumor cells and sickled cells tointegrins expressed in the tumor neovasculature in vivo (see Example 6).Alternatively, the sickled erythrocytes or tumor cells acquire theapolipoprotein or oxyLDL by coculture with liposomes which express theapolipoprotein or oxyLDL (see Section 7 & Example 5).

These tumor cells or sickle cell transfectants are adminsteredparenterally and are capable of trafficking to tumor microvascuaturewherein they bind to apolipoprotein and scavenger receptors onendothelial cells and macrophages. The transfectants are phagocytosed bymacrophages cells and induce endothelial cell apoptosis. SAgs expressedon the tumor cells and sickle cells also induce a local T cellinflammatory anti-tumor response which envelops the neighboring tumorcells.

These tumor cell and sickle cell constructs are prepared by methodsgiven in Examples 1 & 6 and are useful in vivo against primary and/ormetastatic tumors according to Examples 14, 15, 16, 18-23.

Tumor Cells & Endothelial Transfected in vivo with SAg and LipoproteinReceptors or Oxidized Lipoprotein Receptors

The genes encoding the LDL oxyLDL, VLDL, LRP, CD36, SREC and LOX-1receptors as well as macrophage scavenger receptors, expressed onendothelial cells and macrophages and have been cloned. Nucleic acidsencoding receptors for various apolipoproteins including but not limitedto the LDL or apo a, apoB or apo E receptor, CD36 receptor, LRPreceptor, macrophage scavenger receptor, endothelial cell oxyLDLreceptor (LOX-1) and endothelial cell scavenger cell receptor (SREC)alone or together with nucleic acids encoding superantigens are injecteddirectly into tumor sites. The same nucleic acids are transfected intotumor cells in vivo. Transfection of these receptors into tumor cellsand tumor microvascular endothelial cells results in the expression ofthe LDL receptor protein with high affinity binding specificity for LDLoxyLDL and Lp(a).

Exposure of the transfected tumor cells or endothelial cells toexogenously introduced oxidized LDL (especially sterol andlysocholinephosphatidic acid) induces tumor endothelial cell apoptosisanalogous to that seen in endothelial cell after exposure to oxyLDL. Thetransfected tumor cells internalize and degrade the oxyLDL and becausethey, like macrophages, have no means of down regulating the scavengerreceptor are transformed to “foam cells” and undergo apoptosis.

LDL Receptor (LDL-R)

The high affinity receptor for LDL known as the apoB receptor or the LDLreceptor (LDL-R) found on tumor microvascular cells as well as hepaticcells and macrophages binds LDL, VLDL and chylomicron remnants via theirassociated apoproteins. Apolipoprotein B-100 gene has been cloned (ChenS H J. Biol Chem. 261: 12918-12921 (1986)). The LDL gene is more than 45kilobases in length and contains 18 exons. Thirteen of the 18 exonsencode protein sequences that are homologous to sequences in otherproteins: five of these exons encode a sequence similar to one in the C9component of complement; three exons encode a sequence similar to arepeat sequence in the precursor for epidermal growth factor (EGF) andin three proteins of the blood clotting system (factor IX, factor X, andprotein C); and five other exons encode nonrepeated sequences that areshared only with the EGF precursor. The LDL receptor appears to be amosaic protein built up of exons shared with different proteins, and ittherefore belongs to several supergene families (Sudhof T C et al.,Science 228: 815-22 (1985)).

Regulation of LDL-R expression occurs primarily at the transcriptionallevel and is controlled by levels of free cholesterol in the cell.Inflammatory mediators such as growth factors and cytokines can promotethe binding and uptake of LDL. These mediators include PDGF, TGF-b,basic fibroblast growth factor, TNFa, and IL-1. Some of these mediators,such as TNF-a and IL-1, affect transcriptional regulation of the LDL-Rgene at the level of the promoter.

VLDL Receptor

The VLDL receptor has been described as a new member of the LDL receptorsupergene family that specifically binds VLDL and chylomicron remnantsvia apolipoprotein E and lipoprotein lipase. Both apolipoprotein E andlipoprotein lipase are constituents of chylomicron remnants, and aphysiological ligand for the VLDL receptor (Niemeier A et al., J. LipidRes. 37: 1733-42 (1996)).

LRP Receptor

The alpha 2-macroglobulin receptor or lipoprotein receptor-relatedprotein (LRP) (LRP) is a cell-surface glycoprotein of 4525 amino acidsthat functions as a multifunctional receptor which binds and rapidlyinternalizes several plasma proteins. These include alpha2-macroglobulin-protease complexes, free plasminogen activators as wellas plasminogen activators complexed with their inhibitors, andbeta-migrating very low density lipoproteins complexed with eitherapolipoprotein E or lipoprotein lipase tissue and urokinase-typeplasminogen activators, plasminogen activator inhibitor-1, lipoproteinlipase, and lactoferrin. The active receptor protein is derived from a600-kDa precursor, encoded by a 15-kb mRNA, cloned and sequenced inhuman, mouse, and chicken. The entire human gene (LRP1) coding forA2MR/LRP has been cloned. The gene covers about 92 kb and a total of 89exons, varying in size from 65 bases (exon 86) to 925 bases (exon 89)have been identified. The introns vary from 82 bases (intron 53) toabout 8 kb (intron 6). In the introns, 3 complete and 4 partial Alusequences have been identified. Interexon PCR from exon 43 to 45 yieldeda fragment of 2.5 kb. Attempts to subclone this fragment yielded insertsranging between 0.8 and 1.6 kb. Sequencing of 3 subclones withdifferent-size inserts revealed a complex repetitive element with adifferent size in each subclone. In the mouse LRP gene this intron ismuch smaller, and no repetitive sequence was observed. In 18 unrelatedindividuals no difference in size was observed when analyzed byinterexon PCR (Van leuven, F et al., Genomics 24: 78-89 (1994))

The LRP receptor is mainly responsible for the binding andinternalization of chylomicron remnants as well as apoE-containing HDL.ApoE-containing lipoproteins are taken up and degraded byreceptor-mediated endocytosis. Apolipoprotein E3- and apoE4-containinglipoproteins have a similar binding affinity and cause a similar degreeof lipoprotein internalization via the LDL-R and the LRP. LRP canmediate the degradation of tissue factor pathway inhibitor (TFPI), aKunitz-type plasma serine protease inhibitor that regulates tissuefactor-induced blood coagulation

The 3 9-kDa receptor-associated protein (RAP) associates with themultifunctional low density lipoprotein (LDL) receptor-related protein(LRP) and thereby prevents the binding of all known ligands, includingalpha 2-macroglobulin and chylomicron remnants. RAP is predominantlylocalized in the endoplasmic reticulum and functions as a chaperone orescort protein in the biosynthesis or intracellular transport of LRP.RAP promotes the expression of functional LRP in vivo and stabilizes LRPwithin the secretory pathway.

Macrophage Scavenger Receptors

Scavenger receptors mediate the endocytosis of chemically modifiedlipoproteins, such as acetylated low density lipoprotein (Ac-LDL) andoxyLDL. Functional MSR are trimers of two C-terminally differentsubunits that contain six functional domains. The MSR gene has beencloned in an 80-kilobase human and localized to band p22 on chromosome 8by fluorescent in situ hybridization and by genetic linkage using threecommon restriction fragment length polymorphisms. The human MSR geneconsists of 11 exons, and two types of mRNAs are generated byalternative splicing from exon 8 to either exon 9 (type II) or to exons10 and 11 (type I). The promoter has a 23-base pair inverted repeat withhomology to the T cell element. Exon 1 encodes the S-untranslated regionfollowed by a 12-kilobase intron which separates the transcriptioninitiation and the translation initiation sites. Exon 2 encodes acytoplasmic domain, exon 3, a transmembrane domain, exons 4 and 5, analpha-helical coiled-coil, and exons 6-8, a collagen-like domain. Theposition of the gap in the coiled coil structure corresponds to thejunction of exons 4 and 5. The human MSR gene consists of a Macrophagescavenger receptors (MSR) mediate the binding, internalization, andprocessing of a wide range of negatively charged macromolecules.Functional MSR are trimers of two C-terminally different subunits thatcontain six functional domains. The MSR gene has been cloned in an80-kilobase human and localized to band p22 on chromosome 8 byfluorescent in situ hybridization and by genetic linkage using threecommon restriction fragment length polymorphisms. The human MSR geneconsists of 11 exons, and two types of mRNAs are generated byalternative splicing from exon 8 to either exon 9 (type II) or to exons10 and 11 (type I). The promoter has a 23-base pair inverted repeat withhomology to the T cell element. Exon 1 encodes the S-untranslated regionfollowed by a 1 2-kilobase intron which separates the transcriptioninitiation and the translation initiation sites. Exon 2 encodes acytoplasmic domain, exon 3, a transmembrane domain, exons 4 and 5, analpha-helical coiled-coil, and exons 6-8, a collagen-like domain. Theposition of the gap in the coiled coil structure corresponds to thejunction of exons 4 and 5. The human MSR gene consists of a mosaic ofexons that encodes the functional domains. Furthermore, the specificarrangement of exons played a role in determining the structuralcharacteristics of functional domains (Emi M et al., J. Biol. Chem. 268:2120-5 (1993)).

Scavenger receptors on tumor endothelium and stroma bind oxidized LDL,apoptotic cells, and anionic phospholipids. Class A receptors, includesthe type I and II macrophage scavenger receptors (SR-M and SR-MI). Theyare found predominantly on macrophages and activated smooth musclecells. SR-M and SR-MI are homotrimeric membrane proteins, which arederived from alternatively spliced MRNA products of a single gene.Ligands for class A receptors include acetylated LDL, oxidized LDL,fucoidan, and carrageenan. The second class, Class B scavengerreceptors, includes CD36 and SR-E1, which are found in adipose tissue,lung, liver, and macrophages.

Acetyl LDL Receptor

Acetyl LDL receptor or the scavenger receptor, is distinct from the LDLreceptor and does not recognize native LDL. It has been found on tumormicrovascular cells as well as monocyte/macrophages, Kupfer's cells, andendothelial cells, particularly the sinusoidal endothelial cells in theliver. The same receptor also recognizes other chemically modified formsof LDL, including acetoacetyl LDL and malondialdehyde-conjugated LDL.The acetyl LDL receptor binds OxLDL LDL modified by incubation withcultured endothelial cells. LDL incubated with cultured endothelialcells for 12 to 18 hours, undergoes a physical and chemical changes andthe resulting endothelial cell-modified form of LDL is taken up bycultured macrophages 10 times more rapidly than native LDL. Thus, allthree the major cell types in the artery wall can convert LDL to a formrecognized by the acetyl LDL receptor.

CD36 Receptor

CD36, a multigland glycoprotein structurally related to SR-BI and CLA-1found on monocytes, endothelial cells is a high affinity receptor forthe native lipoproteins HDL, LDL, VLDL and for OxLDL and AcLDL. The CD36gene has been cloned (Endemann G et al., J. Biol. Chem. 268:11811-6(1993)).

Endothelial Receptors for OxyLDL: The LOX-1 Receptor (C-Type LectinReceptor)& Scavenger Receptor Expressed by Endothelial Cells (SREC)

Endothelial dysfunction or activation elicited by oxidatively modifiedlow-density lipoprotein (Ox-LDL) is characterized by intimal thickeningand lipid deposition in the arteries. Ox-LDL and its lipid constituentsimpair endothelial production of nitric oxide, and induce theendothelial expression of leukocyte adhesion molecules and smooth-musclegrowth factors. Vascular endothelial cells in culture and in vivointernalize and degrade Ox-LDL through a putative receptor-mediatedpathway that does not involve macrophage scavenger receptors.

LOX-1 Receptor

LOX-1, a novel receptor for oxy-LDL, is a membrane protein that belongsstructurally to the C-type lectin family, and is expressed in vivo invascular endothelium and vascular-rich organs. The LOX-1 receptor fromvascular endothelial cells has been cloned (Hoshikawa H et al., Biochem.Biophys. Res. Commun. 245: 841-6 (1998)). Mouse LOX-1 is composed of 363amino acids with a C-type lectin domain type II membrane proteinstructure and triple repeats of the sequence in the extracellular “Neckdomain,” which is unlike human and bovine LOX-1. LOX-1 binds oxidizedLDL with two classes of binding affinity in the presence of serum. Thebinding component with the higher affinity showed the lowest value of Kdamong the known receptors for oxidized LDL. With respect to ligandspecificity, LOX-1 is a receptor for oxy-LDL but not for Ac-LDL andrecognizes a protein moiety of oxy-LDL with a ligand specificity that isdistinct from other receptors for oxy-LDL, including class A and Bscavenger receptors.

Scavenger Receptor Expressed by Endothelial Cells (SREC),

The primary structure of the SREC molecule has no significant homologyto other types of scavenger receptors, including the LOX-1 receptor. ThecDNA encodes a protein of 830 amino acids with a calculated molecularmass of 85, 735 Da (mature peptide). The cloned has an N-terminalextracellular domain with five epidermal growth factor-like cysteinepattern signatures and long C-terminal cytoplasmic domain (391 aminoacids) composed of a Ser/Pro-rich region followed by a Gly-rich region(Adachi H et al., J. Biol. Chem. 272:31217-20 (1997)

The SREC mediates the binding and degradation of acetoacetylated (AcAc)and acetylated (Ac) low density lipoproteins (LDL). Isolated sinusoidalendothelial cells from the rat liver show saturable, high affinitybinding of AcAc LDL and degrade AcAc LDL 10 times more effectively thanaortic endothelial cells. Specific sinusoidal endothelial cells bearingthe SREC not the macrophages of the reticuloendothelial system, areprimarily responsible for the removal of these modified lipoproteinsfrom the circulation in vivo. For this reason, the SREC receptor and theLOX-1 receptors are preferred for use in transfecting tumor cells tumorendothelium in vivo.

Polypeptide or naked DNA encoding receptors for LDL oxyLDL, VLDL, LRP,CD36, SREC, LOX-1 and macrophage scavenger receptor (collectively o-LDLreceptors) are used individually or together with SAg polypeptidee ornaked DNA containing the CpG backbone are prepared as in Examples 1, 2,3, 30-31. Alternatively, SAg are incorporated or bound or conjugated tovesicular or exosomal structures shed from cells expressing the LDL, oxyLDL receptors. Superantigens are also incorporated into liposomalstructures which express natural or synthetic LDL, oxyLDL receptors asdescribed in Section 45 and Examples 3, 5, 6, 36, 42. All of theseconstructs are administered in vivo by any route but preferably byintratumoral injection as in Examples 2, 6, 14, 30-31. Once localized,and expressing o-LDL receptor(s) in tumor sites in vivo, lipoproteinpreparation(s) containing their respective ligands are administered tothe host. These LDL, oxyLDL or lipoproteins are non toxic to the hostgenerally but upon binding to a dense populaton of receptors in thetumor induce apoptosis of tumor cells and endothelial cells expressingthe receptors and initiate a well localized anti-tumor response. Thepresence of the SAg at the same site amplifies the immune andinflammatory anti-tumor effect. The advantage of this system is theminimal toxicity to the host since the o-LDL receptors are of hostorigin and the lipid infusions consist of substances which areindigenous to the host. These constructs are useful in vivo againstprimary or metastatic tumors according to Examples 14, 15, 16, 18-23.

50. SAg Combined with Tumor Viruses (Nucleic Acid or Peptide Forms)

SAgs are chemically conjugated to HPV-E6 or 7 human papilloma virustumor antigens by methods given in Examples 3. Alternatively, the nakednucleotides containing immunostimulatory sequence of the superantigenand the HPV-E6 or E7 are prepared individually or as a fusion nucleotideor protein as in Examples 5, 30, 31. Alternatively, the the SAg-HPVfusion gene is transfected into tumor cells as given in Example 1. Inthis case, the virus serves as the vector for tranfecting the cells withthe superantigen nucleic acids. The superantigen-HPV-E6 or E7conjugates, fusion proteins, naked DNA fusions or tumor cells expressingthe superantigens and HPV are used as preventative or therapeuticvaccines under protocols given in Examples 14, 15, 16, 18-23, 30, 31.Further, SAg and HPV-E6 or E7 transfected tumor cells are subjected toirradiation or other apoptosis inducing agents or stimuli arter whichthe apoptotic tumor cell transfectants are presented to dendritic cellsex vivo which ingest the apoptotic tumor cells. of In the dendriticcells, the viral antigens and superantigen undergo cross priming to theclass I pathway and these dendritic cells are then harvested andadministered to the tumor bearing host as given in Examples 26-28. TheDNA and RNA from these SAg and HPV-E6 or E7 transfected tumor cells ordendritic cells is extracted and utilized for in vivo therapy as inExamples 30-34. While the HPV-E7 is exemplified herein, the method isapplicable to other viruses which are known to be associated oretiopathogenic in the malignant state including but not limited toadenovirus, EB virus, herpesvirus, hepatitis B, cytomegalovirus andKaposi's sarcoma herpesvirus.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention, unless specified.

EXAMPLE 1 Preparation of Plasmids for Making DNA Templates for any Geneof Interest and the Process Transfection

Mammalian oncogenes, and genes for oncogenic transcription factors,angiogenic factors, growth factor receptors and amplicons as well asbacterial and SAg plasmids and DNA are prepared as described in the textreferences. When necessary, they are modified to forms suitable fortransfection into mammalian tumor cells or accessory cells using methodswell described in the art. (Old R W et al., Principles of GeneManipulation, 5th Ed., Blackwell 1994).

As a representative SAg, enterotoxin B plasmid DNA is prepared by themethod of Jones C L et al., J. Bacteriology 166 29-33 (1986) and Ranelliet al., Proc. Natl. Acad. Sci. USA 82:5850-5854 (1985) using theCsCl-ethidium bromide density gradient centrifugation of cleared lysatesas described (Clewell, D B et al., Proc. Natl. Acad. Sci. USA62-1159-1166 (1969)). S. aureus chromosomal DNA was isolated asdescribed by Betley M et al., Proc. Natl. Acad. Sci. USA 81: 5179-5183(1984). E. coli HB101 was transformed with plasmid DNA by the CaCl₂procedure of Morrison D A et al., Meth. Enzymol. 68:326-331 (1979).Restriction digests were analyzed by 1% agarose and 5% acrylamide gelelectrophoresis using Tris/Borate/EDTA buffer as described in Greene P Jet al., Methods Mol. Biol. 7: 87-111 (1974). Additional methods forisolation and cloning of specific bacterial and mammalian plasmid DNAuseful in tumor or accessory cell transfection are cited in referencesgiven previously in the text or in Snyder L et al., Molecular Geneticsof Bacteria, ASM Press, Washington, D.C.(1997); Peters et al., supra;Franks et al., supra.

Suitable template DNA for production of mRNA encoding a desiredpolypeptide may be prepared using standard recombinant DNA methodologyas described in Ausubel F et al. Short Protocols in Molecular Biology3rd Ed. John Wiley, New York, N.Y. (1995). There are numerous availablecloning vectors and any cDNA containing an initiation codon can beintroduced into the selected plasmid and mRNA can be prepared from theresulting template DNA. The plasmid can be cut with an appropriaterestriction enzyme to insert any desired cDNA coding for a polypeptideof interest. For example the readily available cloning vector pSP64T canbe used after linearization and transcription with SP6 RNA polymerase.Smaller sequence may be inserted into the Hind III/EcoTI fragment withT4 ligase. Resulting plasmids are screened for orientation andtransformed into E. coli. These plasmids are adapted to receive any geneof interest at a unique BglII restriction site which is placed betweenthe two Xenopus □-globin sequences.

Subcloning of SEB into pHb-Apr-1-neo Expression Vector

The Staphylococcal enterotoxin B (SEB) gene has been subcloned intopH□-Apr-1-neo expression vector. The final construct contained only thecoding sequence of SEB and conferred resistance to ampicillin and G-418.

Materials and Methods

PCR:

-   1. The following two primers are designed and made at Life    Technologies, Inc.:    -   PrimerSEB1: total 24 bp 5′ to 3′ GGC.GTC.GAC.ATG.TAT.AAG.AGA.TTA    -   SalI site    -   Primer SEB2: total 24 bp 5′ to 3′        GCC.GGA.TCC.TCA.CTT.TTT.CTT.TGT    -   BamHI site    -   Both primers were dissolved in filter-sterilized ddH₂O to a        final concentration of 20 mM (stock solution).

2. The volume (in ml) of reagents for each PCR reaction is listed below:Reagent Exp. 1 Exp. 2 Exp. 3 Exp. 4 Exp. 5 ddH₂O 76 72 67 49 59 10 × PCRbuffer 10 10 10 10 10 10 × dNTP (2 mM stock) 10 10 10 10 10 Primer SEB11 5 1 10 10 (20 mM stock) Primer SEB2 1 1 1 10 10 (20 mM stock) SEBTemplate 1 1 10 10 0 (50 mg stock) PfuTurbo Enz 1 1 1 1 1 Final Volume100 100 100 100 100

3. The following cycling parameters were applied: 95° C. 1 minute 1cycle initial denature 95° C. 45 seconds denature 52° C. 1 minute 20cycles anneal 72° C. 1 minute extension 72° C. 1 minute 1 cycle finalextension  4° C. hold

-   4. To verify that the PCR reactions yielded the correct size    fragment, 10 ml of the reaction mixture was electrophoresed on a 1%    agarose gel in 1×TAE buffer.    Vector-   1. The pHb-Apr-1-neo expression vector was spotted the vector on a    filter paper. See FIG. 1-   2. To recover the DNA, the circle was cut out and added to 100 ml of    H₂O to allow rehydration for 5 minutes. After a brief    centrifugation, the supernatant was used to transform E. coli    XL1Blue (Stratagene), and selected by ampicillin (final    concentration 100 mg/ml).-   3. To verify that the vector is correct, 4 ampR clones were randomly    selected and the clones were cultured in LB amp media. DNA was    isolated and digested with SalI, BAmHI (single digest) and    EcoRI/HindIII (double digest). The digested products were    electrophoresed on a 1% agarose gel in 1×TAE buffer. The profile of    the restriction digest confirmed that the vector is correct.    Cloning and Verification    -   1. The correct PCR fragments in experiments 2, 3, and 4 were        pooled and gel-purified. A portion of the fragments was digested        with restriction enzymes SalI and BamHi, and was ligated into        the digested pHb-Apr-1-neo expression vector.

The ligation products were transformed into E. coli XL1Blue(Stratagene). Insert containing clones were selected by ampicillin.

-   -   2. Ten ampicillin resistant clones were randomly selected,        cultured in 5 ml of LB amp media, and their plasmid DNA was        isolated. Insert containing clones (SEB construct) were verified        by digesting the DNA with SalI and BamHI restriction        endonucleases and electrophoresis at 0.8% agarose gel. (FIG. 2)    -   3. One of the SEB constructs (clone #2) was verified by        sequencing and aligned with the published SEB sequence (FIG. 3).    -    Purified DNA templates from bacteria and human cells are        prepared for introduction of plasmid into human and bacterial        cells by additional methods given in Ausubel F et al., supra.        The plasmid DNA is grown up in E. coli in ampicillin containing        LB medium. The cells were then pelleted by spinning a 5000 rpm        for 10 min. at 5000 rpm., resuspended in cold TE pH 8.0,        centrifuged again for 10 minutes. at 5000 rpm., resuspended in a        solution of 50 mM glucose, 25 mM Tris-Cl pH 8.0, 10 mM EDTA and        40 mg/ml lysozyme. After incubation for 5-10 min. with        occasional inversion, 0.2 N NaOH containing 1% SDS was added,        followed after 10 minutes at 0° C. with 3 M potassium acetate        and 2 M acetic acid. After 10 more minutes, the material was        again centrifuged a 6000 rpm, and the supernatant was removed        with a pipet. The pellet was then mixed into 0.6 vol.        isopropanol (−20° C.), mixed, and stored at −20° C. for 15        minutes. The material was then centrifuged again at 10,000 rpm        for 20 min., this time in an HB4 singing bucket rotor apparatus        after which the supernatant was removed and the pellet was        washed in 70% EtOH and dried at room temperature. Next, the        pellet was resuspended in 3.5 ml TE, followed by addition of 3.4        g CsCl and 350 l of 5 mg/ml EtBr. The resulting material was        placed in a quick seal tube, filled to the top with mineral oil.        The tube was spun for 3.5 hours at 80,000 rpm in a VTi80        centrifuge. The band was removed and the material was        centrifuged again making up the volume with 0.95 g CsCl/ml and        0.1 ml or 5 mg/ml EtBr/ml in TE. The EtBr was then extracted        with an equal volume of TE saturated N-Butanol after adding 3        volumes of TE to the band. Next, 2.5 vol. EtOH was added, and        the material was precipitated at −20° C. for 2 hours. The        resultant DNA precipitate is used as a DNA template.        Transfection of B16F10 Melanoma Cells

G418 sensitivity: B16F10 melanoma cells (B16s) were first tested forsensitivity to G418 which will be used as the selectable marker. At 400ug/mL G418, B 16s did not survive, while 200 and 300 ug/mL allowed somesurvival.

Transfection:

Lipofectamine was used to produce stably transfected B16s. Theconditions for transfection were those described protocol provided byLife Technologies. B16s were plated at 4×105 cells/well in 6 wellplates, using Murine Complete Medium (MCM) described in Report 2. Cellswere cultured overnight. Optimal density is 50-80% confluent and isusually achieved by 18-24 after seeding at 1-3×105 cells/well. DNAsources consisted of SEB-G418 resistance containing vector, vector DNAwith G418 resistance gene only, and control DNA from PSK401 (no G418resistance marker). DNA concentrations were determined for the SEBcontaining and control vectors. DNA source A260 DNA (ug/ml) SEB 0.090.45 Vector only 0.13 0.65 PSK 401 0.15 0.75

Lipofectamine solutions and DNA solutions were prepared in 12×75 mmtubes, using OPTI-MEM (Life Technologiies 31985). DNA solutionscontained approximately 2 ug in 100 uL OPTI-MEM; the LIPOFECTAMINEReagent was diluted by adding 6 or 12 uL to OPTI-MEM at a final volumeof 100 uL. The solutions were mixed and held at room temperature for 30minutes. Specific DNA and Lipofectamine conditions were as follows:

Plated cells were rinsed once with 2 ml/well OPTI-MEM. To the abovetubes, 0.8 mL OPTI-MEM. This mixture was then overlayed onto the washedcell monolayers according to the above well designations. Cells wereincubated for 5 hours at 37° C. in 5% CO2. Murine Complete Medium with20% FBS but no antibiotics was then added at 1 ml/well. Cultures wererefed with standard MCM, at 3 mL/well, after 24 hours. Three days aftertransfection, cells from each transfection condition were subcultured bysplitting the total cell suspension 90:10 into 150 mm plates (one platereceived 90% of the cell suspension, the other received the remaining10%).

G418 Selection

All plates were refed at 6 days after transfection with mediumcontaining 400 ug/mL G418. Plates were refed every 2 to 3 days with G418containing medium until day 17 after transfection. No growth wasobserved in wells 1-4 as expected. Plates initiated with 90% of the cellsuspension and showing growth were harvested, frozen, and stored at −80°C.

Primary Subcloning

Ten colonies were selected from each well for wells 5, 7, 9, and 11.Subcloning was accomplished by the use of cloning cylinders as follows:After seating the cylinder, medium was aspirated and the isolated colonywas washed once with 100 uL of warmed trypsin-EDTA. This was aspiratedand replaced with fresh tyrpsin-EDTA. After incubation at 37° C. for 2minutes, the cells were recovered by trituration and transferred to atube containing 1 ml MCM, then replated by addition of 20 uL of cellsuspension to 15 mL MCM with G418 in 150 mm plates. The remaining cellsuspension was plated into 24 well plates, 4 wells/clone and all plateswere maintained at 37° C., 5% CO2. The 6 well plates were used to assessSEB expression on the cell surface as desribed under Detection ofpositive clones.

Secondary and Tertiary Subcloning and Preparation of Frozen Stocks

These and all subsequent procedures were performed by me. Secondarysuncloning was performed as above at 7 days after initiation of primarysubclones. One colony/plate was selected for further subcloning (a totalof 40 colonies) The cell suspension was prepared in a total volume of 1mL; 100 uL was replated into 100 mm plates containing 10 mL MCM withG418. The remaining cell suspension was plated in 96 well plates at100/well, 2 replicates for assay. The 96 well plate was used fordetection of intracellular expression of SEB desribed under Detection ofpositive clones.

Primary subcloning plates were cultured one additional day, thenharvested, frozen, and stored at −80° C. These frozen stocks aredesignated primary subclones. Secondary subclones were refed after 4days. Of 40 secondary clones, 36 regrew. Tertiary subcloning wasperformed after 8 days and frozen stocks of secondary clones wereprepared after 9 days. Tertiary clones were refed after 3 days inculture and subcultured after 7 days in culture. Plates were harvested,cells were resuspended in a total of 1 mL, and replated by addition of100 uL of the cell suspension to 100 mm plates with 15 mL MCM or 100uL/well in a 96 well plate. Frozen stocks of tertiary clones wereprepared.

Generation of Conditioned Medium for Assay of Supernatents

After 7 days, 100 mm plates of tertiary clones were again replated. Thistime, cell counts were performed and 4.5×10⁵ cells were plated in 12well plates, one well/clone. The remaining cell suspension was frozenand stored at −80° C. After 4 days in culture, supernatents wereharvested, stored at 4° C., and the cells were replated into 100 mmplates. Supernatents were obtained from the 100 mm plates after 7 daysin culture. See Detection of positive clones. Frozen stocks were alsogenerated from these plates.

Development of ELISA with HRP Rabbit anti-SEB.

Final ELISA conditions were as follows: Assay Plate ProBind (Falcon#3915) Capture Antibody Rabbit anti-SEB (Toxin Technologies #LBI202), 10ug/mL in PBS, 50 uL/well, 1 hr, RT Wash 3 × with 0.1% casein, 0.1% Tween20 in PBS Blocking 1% casein in PBS, 250 uL/well, overnight, 4° C.Antigen Supernatant used neat or SEB diluted in PBS, 50 uL/well, 2 hr,RT Wash As above Primary Ab HRP Rabbit anti-SEB (Toxin Technologies #LBC202), 1/300 in block buffer, 50 uL/well, 2 hr, RT Substrate OPD, 2.5mg/mL in citrate buffer, pH 5.0, 0.03% H₂0₂, 100 ul/well, 15 min, RTStop 4 M H2SO4, 100 uL/well Read-out OD 490 nm

Results: SEB produced a dose response curve (linear range 60 fg−60pg/mL) and the background was very low. Vector only clones produced onlybackground, signals. One SEB transfected clone produced a strong signal,three produced moderate signals, and one other produced a weak butdefinite signal. OD 490 nm SEB+ Vector only 1 2 mean 1 2 mean 9.1 0.0970.112 0.104 0.079 0.102 0.091 9.2 0.127 0.123 0.125 0.081 0.076 0.0789.3 0.109 0.104 0.106 0.087 0.070 0.079 9.4 0.444 0.393 0.418 0.0770.077 0.077 9.5 0.163 0.087 0.125 0.075 0.074 0.074 9.6 0.516 0.5220.519 0.066 0.064 0.065 9.7 0.087 0.091 0.089 0.096 0.084 0.090 9.80.386 0.450 0.418 0.080 0.071 0.075 9.9 0.137 0.122 0.130 0.071 0.0700.071 11.1 0.083 0.075 0.079 0.068 0.078 0.073 11.2 1.847 1.802 1.8240.063 0.076 0.070 11.3 0.071 0.077 0.074 0.076 0.074 0.075 11.4 0.0870.084 0.086 0.083 0.085 0.084 11.5 0.161 0.220 0.191 0.092 0.086 0.08911.8 0.221 0.100 0.160 0.080 0.081 0.080 11.9 0.080 0.091 0.085 0.0770.072 0.074 11.10 0.290 0.254 0.272 0.081 0.112 0.097 11.10 0.268 0.2630.265 0.093 0.114 0.103

Based on the the SEB standard curve, the following concentrations werederived. Clone number(pg/ml) SEB 11.2 4.146 9.6 0.152 9.4 0.118 9.80.118 11.10 0.081

Cells are transfected ex vivo or in vivo and implanted in acancer-bearing host. These transfected cells are also used to stimulatehost lymphocytes ex vivo. Once activated, the lymphocytes areadministered to the host. The ex vivo or in vitro introduction of DNAinto cells is accomplished by methods that (1) form DNA precipitateswhich are internalized by the target cell; (2) create DNA-containingcomplexes with charge characteristics that are compatible with DNAuptake by a target cell; or (3) result in the transient formation ofpores in the plasma membrane of a target cell exposed to an electricpulse (these pores are of sufficient size to allow DNA to enter thetarget cell).

Generally, two factors determine the method used: the duration ofexpression required (i.e., transient versus stable expression) and thetype of cell to be transfected. The specific details of exemplaryprocedures are described herein.

Transfections are carried out by well established methods includingcalcium phosphate precipitations, DEAE Dextran transfection, andelectroporation.

Calcium Phosphate Precipitation

A commonly used ex vivo and in vitro method to transfer DNA intorecipient cells involves the co-precipitation of the DNA of interestwith calcium phosphate. With this technique, DNA enters the cell insufficient quantities such that the treated cells are transformed withrelatively high frequency. Using a variety of cell types, transfectionefficiencies of up to 10-3 have been obtained. This is the method ofchoice for the generation of stable transfectants.

Variations of the basic technique have been developed. If thetransfection involves the transfer of plasmid DNA, then high molecularweight genomic DNA isolated from a defined cell or tissue source can beincluded. The addition of such DNA, called carrier DNA, often increasesthe efficiency of transfection by the plasmid DNA. Upon arrival of theplasmid DNA/carrier DNA/calcium phosphate co-precipitate to the nucleusof the treated cell, the plasmid DNA integrates into the carrier DNA,often in the tandem array, and this assembly of plasmid and carrier DNA,called a transgenome, subsequently integrates into the chromosome of thehost cell.

Another procedural option is the addition of a chemical shock step tothe transfection protocol. Either dimethylsulfoxide or glycerol areappropriate. The optimal concentrations and lengths of treatment varyaccording to cell type. The use of these agents dramatically affect cellviability and can be optimized as described elsewhere [Chen and Okayama,Mol. Cell. Biol. 7:2745 (1987)]. Specifically, incubation of cells withthe co-precipitate is optimal at 35° C. in 2-4% CO₂ for 15-24 hours. Inaddition, circular DNA is more active than linear DNA and a finerprecipitate is obtained when the DNA concentration is between 20-30mg/ml in the precipitation mix.

It is noted that incubator temperature, CO₂ concentration, and DNAconcentration can be varied to obtain the desired result. In addition,the temperature and CO₂ concentrations described below are not optimalfor cell growth and should be maintained only temporarily.

Method

-   Day 1: 1.3×10⁶ cells are seeded per 100-mm dish. Cells are about 75%    confluent when used to seed the dishes.-   Day 2: A large calcium phosphate cocktail mixture to transfect many    plates simultaneously is prepared. This protocol is given for 1 ml    (or 1×100-mm dish equivalent) of solution. These amounts are scaled    up as necessary, allowing for an appropriate amount of    sample-transfer errors. Adherence to sterile technique is critical.    Sterile reagents, tips, and tubes are used.    -   1. Add 1-20 g DNA (1 mg/ml in sterile TE, 10 mM Tris-HCl 1 mM        EDTA pH 7.05) to 0.45 ml sterile H₂O. Note: First “sterilize”        DNA by ethanol precipitation with NaCl (0.15293 M final aqueous        concentration) and 2× volume 200% ethanol.    -   2. Add 0.5 ml 2×HEPES buffered saline. Mix well.    -   3. Add 50 ml of 2.5 M CaCl₂, vortex immediately.    -   4. Allow the DNA mixture to sit undisturbed for 15-30 minutes at        room temperature.    -   5. Add 1 ml of the DNA transfection cocktail directly to the        medium in the 100-mm dish (plated with cells on day 1).    -   6. Incubate the dishes containing the DNA precipitate for 16        hours at 37° C. Remove the media containing the precipitate and        add fresh complete growth media.    -   7. Allow the cells to incubate for 24 hours. Post-incubation,        the cultures can be split for subsequent selection. Split        cultures 1:5; however, to isolate individual colonies for        further analysis, split cultures 1:10 and 1:100.        DEAE Dextran Transfection

Typically, DEAE dextran transfection is used to transiently transfectcells in culture. This method is highly efficient and the DNA/DEAEdextran mixture used for transfection is relatively easy to prepare. Forexample, this method yields transfection efficiencies of as high as 80percent. DNA introduced into cells with this method, however, appears toundergo mutations at a higher rate than that observed with calciumphosphate-mediated transfection.

Method

Briefly, a DEAE dextran mixture is prepared and the DNA sample ofinterest is added, mixed, and then transferred to the cells in culture.

-   Day 1: Cells are seeded at a concentration of 2×10⁴ cells/cm2 in a    total volume of 2 ml/well (1.92×10⁵ cells/well of a six-well cluster    dish). Cells should be about 75% confluent when used to seed the    dishes.-   Day 2: Resuspend 0.5 ml DEAE Dextran in Tris-buffered saline (TBS).    Final DEAE Dextran concentration should be about 0.04%. Observe cell    monolayers microscopically. Cells should appear about 60-70%    confluent and well distributed. Bring all reagents to room    temperature. Aspirate off growth media and wash monolayer once with    3 ml of phosphate buffered saline (PBS), followed by one wash with 3    ml of TBS. Aspirate off TBS solution and add 100-125 ml of the    appropriate DNA/DEAE-Dextran/TBS mixture to the wells. Incubate    dishes at room temperature inside a laminar flow hood. Rock the    dishes every 5 minutes for 1 hour, making sure the DNA solution    covers the cells. After the 1-hour incubation period, aspirate off    the DNA solution and wash once with 3 ml of TBS followed by 3 ml of    PBS. Remove the PBS solution by aspiration and replace with 2 ml of    complete growth media containing 100 M chloroquine.

Incubate the dishes in an incubator set at 37° C. and 5% CO₂ for 4hours. Remove the media containing chloroquine and replace with 2-3 mlof complete growth media (no chloroquine). Incubate the transfectedcells for 1-3 days, after which the cells will be ready for analysis.The exact incubation period depends on the intent of the transfection.Optimal expression typically occurs at 3days post-transfection.

Electroporation

Electroporation is a process whereby cells in suspension are mixed withthe DNA to be transferred. This cell/DNA mixture is subsequently exposedto a high-voltage electric field. This creates pores in the membranes oftreated cells that are large enough to allow the passage ofmacromolecules such as DNA into the cells. Such DNA molecules areultimately transported to the nucleus and a subset of these moleculesare integrated into the host genome. The reclosing of the membrane poresis both time and temperature dependent and thus is delayed by incubationat 0° C., thereby increasing the probability that the molecule ofinterest will enter the cell.

Electroporation appears to work on virtually every cell type. With thistechnique, the efficiency of nucleic acid transfer is high for bothtransient transfection and stable transfection. One important technicaldifference between electroporation and other competing technologies isthat the number of input cells required for electroporation isconsiderably higher.

Method

-   1. Harvest exponentially growing cells such as tumor cells or    accessory cells by trypsinization, pellet, and wash twice with    electroporation buffer (Kriegler, M. Gene Transfer and Expression,    W.H. Freeman and Co., New York, N.Y. (1991)).-   2. Resuspend cells in electroporation buffer at a concentration of    2-20×10⁶ cells/ml in an electroporation cuvette.-   3. Add 5-25 mg of DNA that has been linearized to the cell    suspension-   4. Insert or connect the electroporation electrode according to the    manufacturer's instructions and subject cell/DNA mixture to an    electric field (pulse).-   5. Return cell/DNA mixture to ice and incubate for 5 minutes.-   6. Plate cells in non-selective medium. Biochemical selection may be    carried out 24-48 hours later.    Lipofectamine

In vitro cell transfections can be done in 12-well plates, using 3.0 gplasmid DNA and Lipofectamine (GIBCO BRL), at 37° C. for 4 hours. Aftertransfection, the cells are cultured in 2.0 ml complete medium for 48hours and the cells are harvested. The cells are then washed in PBS.Stably transfected Chinese hamster ovary (CHO) and B 16 lines areisolated by selection in 1.0 mg/ml G418 (GIBCO BRL). Cells are grown andpassaged in medium containing G418 for 3-4 weeks Mock transfected celllines (cells transfected with vector only) are used as controls.

Viral Vectors

Recombinant viral vectors containing the nucleic acid of interest canalso be used to introduce nucleic acid into a cell ex vivo or in vitro.It is noted that viral vectors are also used to transfect cells in vivo.These viral vectors can be DNA viruses such as herpesviruses,adenoviruses, and vaccinia viruses or RNA viruses such as retroviruses.The method and materials required to produce and use these viral vectorsex vivo, in vitro, and in vivo are commonly known in the art and areused in the invention described herein (Sambrook, J. et al., supra).

Selection

Regardless of the method used to transfect a particular cell type,stably transfected cells are identified as follows. The DNA of interestcontains a selectable marker. Typically, a selectable marker encodes apolypeptide that confers drug resistance and the DNA containing thisresistance conferring nucleic acid is transfected into the recipientcell. Post transfection, the treated cells are allowed to grow for aperiod of time (24-48) hours to allow for efficient expression of theselectable marker. After an appropriate incubation time, transfectedcells are treated with media containing the concentration of drugappropriate for the selective survival and expansion of the transfectedand now drug resistant cells.

Many drug as well as non-drug selection methods are known in the art andcan be used in the invention described herein. For example, a detaileddescription of currently available drug selection strategies is providedin Kriegler M., Gene Transfer and Expression, A Laboratory Manual, W.H.Freeman and Co. New York, N.Y. pp. 103-107 (1991).

General Method

Sixteen hours after transfection, the transfected/infected cells are fedwith fresh, non-selective media. Twenty-four to forty-eight hours later,the cultures are split to a 1:5 or greater dilution and plated indrug-containing media. It is noted that cells are not placed indrug-containing media immediately after transfection in order to allow asufficient amount of time for the drug resistance nucleic acid to beexpressed and thus confer the drug resistant phenotype. Cell culturesare re-fed with drug-containg media every three days, at which timecultures are examined under a microscope to determine the efficiency ofdrug selection.

Site-Directed Mutagenesis by Polymerase Chain Reaction: Introduction ofRestriction Endonuclease Sites by PCR

PCR is the preferred method for introducing any desired sequence changeinto the DNA. The basic protocol is as follows:

Materials

-   DNA sample to be mutagenized, pUC19 plasmid b vector or similar    high-copy number plasmid having M13 flanking primer-   500 ng/ml (100 pM/μl) flanking sequence primers incorporating the    restriction enzyme site-   TE buffer-   10× amplification buffer-   2 mM 4dNTP mix-   500 ng/ml (100 pM/ml) M13 flanking sequence primers: forward (NEB)    and reverse (NEB)-   5 U/ml Taq DNA polymerase-   Mineral oil-   Chloroform-   Buffered phenol-   100% ethanol-   Appropriate restriction endonucleases-   500 ml microcentrifuge tube-   Automated thermal cycler-   1. Subclone DNA to be mutagenized into high-copy number vector using    restriction sites flanking the area to be mutated.-   2. Prepare template DNA by plasmid miniprep. Resuspend 100 ng in TE    buffer to 1 ng/ml final.-   3. Synthesize oligonucleotide primers and purify by denaturing    polyacrylamide gel electrophoresis. Resuspend oligonucleotides in    500 1 TE buffer. Determine absorbance at A260 and adjust to 500    ng/ml.-   4. Combine the following in each of two 500 l microcentrifuge tubes,    adding oligonucleotides 1 and 2 to separate tubes:    -   10 ml (10 ng) template DNA    -   10 ml 10× amplification buffer    -   10 ml 2 mM 4dNTP mix    -   1 ml (500 ng) oligonucleotide 1 or 2 (100 pM final)    -   1 ml (500 ng) appropriate M 13 flanking sequence primer, forward        or reverse (100 pM final).    -   H₂O to 99.5 μl    -   0.5 ml Taq DNA polymerase (5U/ml)    -   Overlay reaction with 100 ml mineral oil.-   5. Carry out PCR in an automated thermal cycler for 20 to 25 cycles    under the following conditions:    -   45 sec 93° C.    -   2 min 50° C.    -   2 min 72° C.    -   After last cycle, extend for an additional 10 min at 72° C.-   6. Analyze 4 l by nondenaturing agarose or occurrence gel    electrophoresis to verify that the amplification has yielded the    predicted product.-   7. Remove mineral oil and extract once with chloroform to remove    remaining oil. Extract with buffered phenol and concentrate by    precipitation with 100% ethanol.-   8. Digest half the amplified DNA with the restriction endonucleases    for the flanking and introduced sites. Purify digested fragments on    a low gelling/melting agarose gel.-   9. Ligate and subclone both fragments into an appropriately digested    vector to obtain a recombinant plasmid containing a single DNA    fragment incorporating the new restriction site.-   10. Transform plasmid into E. coli. Prepare DNA by plasmid miniprep.-   11. Analyze amplified fragment portion of plasmid by DNA sequencing    to confirm the addition of the mutation.    Introduction of Point Mutation by PCR:    -   Materials    -   DNA sample to be mutagenized    -   Oligonucleotide primers incorporating the point mutation    -   Klenow fragment of E. coli DNA polymerase I    -   Appropriate restriction endonuclease    -   Procedure    -   1. Prepare template DNA (steps 1 and 2 of Basic Protocol).    -   2. Synthesize and purify oligonucleotide primers (3 and 4).    -   3. Amplify template DNA (steps 4 and 5 of Basic Protocol 1).        After final extension step, add 5 U Klenow fragment and incubate        15 min at 30° C.).    -   4. Analyze and process reaction (steps 6 and 7 of Basic        Protocol).    -   5. Digest half the amplified fragments with the restriction        endonucleases for the flanking sequences. Purify digested        fragments on a low gelling/melting agarose gel.    -   6. Subclone the two amplified fragments into an appropriately        digested vector by blunt-end ligation.    -   7. Carry out steps 10 and 11 of Basic Protocol.        Introduction of a Point Mutation by Sequential PCR Steps    -   1. Prepare the template DNA (steps 1 and 2 of Basic Protocol 1).    -   2. Synthesize and purify the oligosaccharide primers (5 and 6).    -   3. Amplify the template and generate blunt-end fragments (step 3        of Basic Protocol).    -   4. Purify fragments by nondenaturing agarose gel        electrophoresis. Resuspend in TE buffer at 1 ng/ml.    -   5. Combine the following in 500 ml microcentrifuge tube:        -   10 ml (10 ng) each amplified fragment        -   1 ml (500 ng) each flanking sequence primer (each 100 pM            final)        -   10 ml 10× amplification buffer        -   10 ml 2 mM 4dNTP mix        -   0.5 ml Taq DNA polymerase (5 U/ml)        -   Overlay with 100 ml mineral oil.    -   6. Carry out PCR for 20 to 25 cycles (step 5 of Basic Protocol        1). Analyze and process the reaction mix (steps 6 and 7 of Basic        Protocol 1).    -   7. Digest cDNA fragment with appropriate restriction        endonuclease for the flanking sites. Purify fragment on a low        gelling/melting agarose gel. Subclone into an appropriately        digested vector.    -   8. Carry out steps 10 and 11, Basic Protocol 1.        Genomic Targeting and Genetic Conversion in Cancer Therapy

A number of cellular transformations are due, in large part, to a singlebase mutation that alters the function of the expressed protein.Alterations in the DNA sequence of a gene involved in cell proliferationcan have a significant effect on the viability of particular cells.Thus, the capacity to modulate the base sequence of such a gene would bea useful tool for cancer therapeutics. An experimental strategy thatcenters around site-specific DNA base mutation or correction using aunique chimeric oligonucleotide has been developed. This chimericmolecule has demonstrated higher recombinogenic activities thanidentical oligonucleotides containing only DNA residues, both in vitroand in vivo. The chimeric molecule is designed to hybridize to a targetsite within the genome and induce a single base mismatch at the residuetargeted for mutation. The DNA structure created at this site isrecognized by the host cell's repair system which mediates thecorrection reaction. For example, the bcr-abl fusion gene, the productof a translocation between human chromosomes 9 and 22, and the cause ofchronic myelogenous leukemia (CML) can be targeted for gene correction.Fusion genes or mutations which abound in cancer cells are excellenttargets for correction especially if (1) they are unique and arerecognized by the immune system as dominant or subdominant epitopes, (2)they are a single copy target; (3) the DNA sequence of the fusion geneor mutation is unique. The goal of such experiments is to knock-out thefusion gene by changing an amino acid codon into a stop codon through achimeric directed DNA repair system.

Targeted Gene Correction of Episomal DNA in Mammalian Cells Mediated bya Chimeric RNA/DNA Oligonucleotide

An experimental strategy to facilitate correction of single-basemutations of episomal targets in mammalian cells has been developed. Themethod utilizes a chimeric oligonucleotide composed of a contiguousstretch of RNA and DNA residues in a duplex conformation with doublehairpin caps on the ends. The RNA/DNA sequence is designed to align withthe sequence of the mutant locus and to contain the desired nucleotidechange. Activity of the chimeric molecule in targeted correction is usedin a with the aim of correcting a point mutation in the gene encodingthe human liver/bone/kidney alkaline phosphatase. When the chimericmolecule is introduced into cells containing the mutant gene on anextrachromosomal plasmid, correction of the point mutation isaccomplished with a frequency approaching 30%. These results extend theusefulness of the oligonucleotide-based gene targeting approaches byincreasing specific targeting frequency.

The site directed mutagenesis is used to carry out using the chimericDNA/RNA structure which enables the construct to target tumor cells invivo and in vitro. Such targeting structures include target seekingmoieties and can in principle be any structure that is able to bind to acell surface structure or that binds via biospecific affinity. Thetarget seeking moiety is primarily a disease specific structure selectedamong hormones, antibodies, growth factors. The biospecific affinitycounterpart may include interleukins (especially interleukin-2)antibodies (full length antibody, Fab, F(ab′₂, Fv, single chain antibodyand any other antigen binding antibody fragments (such as Fab) directedto a cells surface epitope or more preferably towards the bindingepitope for the a specific antibody. They may also include polypeptidesbinding to the constant domains of immunoglobulins (e.g., protein A andG and L), lectins, streptavidin, biotin etc. The term antibodiescomprises monoclonal as well as polyclonal preparations. The targetingmoiety may also be directed toward unique structures on more or lesshealthy cells that regulate or control the development of a disease. orligands for specific receptors on tumor cells). The targeting structuremay be a nucleic acid, lipid or carbohydrate and variations thereofwhich target receptors on the diseased cell. The targeting is notconfined to diseased cells but may include additional normal cells aswell.

Synthesis and Purification of Oligonucleotides.

The chimeric oligonucleotides are synthesized on a 0.2-mol scale byusing the 1000 Å-wide-pore CPG on the ABI 394 DNA/RNA synthesizer. Theexocyclic amine groups of DNA phosphoramidites (Applied Biosystems) areprotected with benzoyl for adenosine and cytidine and isobutyryl forguanosine. The 2′-O-methyl RNA phosphoramidites (Glen Research,Sterling, Va.) are protected with a phenoxyacetyl group for adenosine,dimethylformamide for guanosine and an isobutyryl group for cytidine.After the synthesis is complete, the base-protecting groups are removedby heating in ethanol/concentrated ammonium hydroxide, 1:3 (vol/vol),for 20 h at 55° C. The crude oligonucleotides are purified bypolyacrylamide gel electrophoresis. The entire oligonucleotide sample ismixed with 7 M urea/10% (vol/vol) glycerol. heated to 70° C., and loadedon a 10% polyacrylamide gel containing 7 M urea. After gelelectrophoresis, DNA is visualized by UV shadowing, dissected from thegel, crushed, and eluted overnight in TE buffer (10 mM Tris-HCl/1 mMEDTA, pH 7.5) with shaking. The eluent containing gel pieces arecentrifuged through 0.45-um (pore size) spin filter (Millipore) andprecipitated with ethanol. Samples are further desalted with a G-25 spincolumn (Boerhinger Mannheim) and greater than 95% of the purifiedoligonucleotides are found to be full length.

Transient Transfection and Measurements of Activity

CHO cells were maintained in Dulbecco's modified Eagle's medium (DMEM)(BRL) containing 10% (vol/vol) fetal bovine serum (FBS; BRL). Transienttransfection is carried out by addition of 10 g of the plasmid with 10 gof Lipofectin in 1 ml of Optimem (BRL) to 2×10⁵CHO cells in a 6-wellplate. After 6 h. various amounts of oligonucleotide is mixed with 10 gof Lipofectin in 1 ml of Optimem and added to each well. After 18 h, themedium is aspirated and 2 ml of DMEM containing 10% FBS was added toeach well. Histochemical staining was carried out (19), 24 h aftertransfection of the oligonucleotide. Spectrophotometric measurements arecarried out by the ELISA amplification system (BRL). Transfection iscarried out in triplicate in a 96-well plate. The amounts of reagentsand cells are 10% of that used for the 6-well plate. Cells were washedthree times with 0.1 SM NaCl and lysed in 100 μl of buffer containing 10mM NaCl, 0.5 Nonidet P-40, 3 mM MgCl2, and 10 mM Tris-HCl (pH 7.5), 24 hafter transfection with chimeric oligonucleotides. A fraction of celllysates (20 μl) incubated with 50 l of ELISA substrate and 50 μl ofELISA amplifier (BRL), the reaction is stopped by addition of 50 μl of0.3 M H₂S04 after 5 min of incubation with amplifier. The extent ofreaction is carried out within the linear range of the detection method.The absorbance is read by an ELISA plate reader (BRL) at a wavelength of490 nm.

Hirt DNA Isolation, Colony Hybridization and Direct DNA Sequencing ofPCR Fragments

The cells are harvested for vector DNA isolation by a modified alkalinelysis procedure, 24 h after transfection with the chimericoligonucleotide. Hirt DNA is transformed into Escherichia coli DH5acells (BRL). Colonies from Hirt DNA are screened for specifichybridization for each probe designed to distinguish the point mutation.Colonies were grown on ampicillin plates, lifted onto nitrocellulosefilter paper in duplicates, and processed for colony hybridization.

The blots were hybridized to ³²P-end-labeled oligonucleotide probes at37° C. in a solution containing 5× Denhardt's solution, 1% SDS, 2×SSC,and denatured salmon sperm DNA (100 μg/ml). Blots were washed at 52° C.in TMAC solution (3.0 M teramethylammonium chloride/50 mM Tris-HCl, pH8.0/2 mM EDTA/0.1% SDS). Plasmid DNA was made from 20 colonies shown tohybridize to either of the probes by using the Qiagen miniprep kit(Chatsworth. Calif.). Several hundred bases flanking key positions ofeach plasmid are sequenced in both directions by automatic sequencing(ABI 373A, Applied Biosystems). A 192-bp PCR-amplified fragment aregenerated by Vent polymerase (New England Biolabs. MA), utilizingprimers corresponding to positions of the known cDNA flanking position.The fragment is gel-purified and subjected to automatic DNA sequencing(ABI 373A, Applied Biosystems).

Oligonucleotide Synthesis

Chimeric RNA/DNA oligonucleotides for both transcribed andnontranscribed factor IX were synthesized by Applied Biosystems, Inc.(Foster City, Calif.) as previously described. The oligonucleotides areprepared with DNA and 2-O-methyl RNA phosphoramidite nucleoside monomerson an ABI 394 DNA/RNA synthesizer, purified by HPLC and quantified by UVabsorbance. More than 95% of the purified oligonucleotides aredetermined to be full length.

Cell Isolation and Transfections

Cells are isolated, by a two-step collagenase perfusion as previouslydescribed. The purified cells are plated on Primaria plates (BectonDickinson, Franklin Lakes, N.J.) at a density of 4×10⁶ cells per 35-mmdish and maintained in supplemented William's E medium. Eighteen hoursafter plating, the cells are washed and transfected with the chimericmolecules complexed to polyethylenimine (PEI). A pH 7.0, 10 mM stocksolution of PEI (800 kDa) (Fluka Chemical Corp., Ronkonkoma, N.Y.) isprepared. Briefly, the chimeric oligonucleotides are complexed with 10mM PEI at 9 equivalents of PEI nitrogen per chimeric phosphate in 100 1of 0.15 M NaCl and transfected in 1 ml of medium at final concentrationsof 150, 300 or 450 nM. PEI is lactosylated by coupling lactose to 30% ofthe nitrogen amines using sodium cyanoborohydride (Sigma ChemicalCompany, St. Louis, Mo.). Cells are also transfected 1with 100 l of 0.15M NaCl containing the lactosylated 800-kDa and 25-kDa PEI chimericcomplexes (Sigma) at final concentrations of 90, 180 or 270 nM. After 18h, an additional 2 ml of medium is added to the transfected cultures forthe remaining 6 or 30 h of incubation. Vehicle control transfectionsutilize the same amount of PEI, but substituted an equal volume of 10 mMTris-HCl, pH 7.6, for the oligonucleotides.

DNA/RNA Isolation and Cloning

The cells were harvested by scraping 48 h after transfection. GenomicDNA larger than 100-150 base pairs was isolated using the highly purePCR template preparation kit (Boehringer Mannheim, Indianapolis, Ind.).RNA was isolated using RNAzoI 8 (Tel-Test, Inc., Friendswood, Tex.),according to the manufacturer's protocol. PCR amplification of afragment of the gene in question gene is performed with 300 ng of theisolated DNA from either the primary cell culture.

The primers were designed (Oligos Etc., Wilsonville, Oreg.)corresponding to nucleotides to cDNA to be corrected (ref 25). Primerannealing is carried out at 59° C., and the samples are amplified for 30cycles using Expand Hi-fidelity polymerase (Boehringer Mannheim). Torule out PCR artifacts, 300 ng of control DNA is incubated with 0.5, 1.0and 1.5 g of the oligonucleotide before the PCR-amplification reaction.Additionally, 1.0 g of the chimeric alone is used as the “template” forthe PCR amplification.

RT-PCR amplification is done utilizing the Titian one tube RT-PCR system(Boehringer Mannheim) according to the manufacturer's protocol and byusing the same primers as those used for the DNA PCR amplification. Torule out DNA contamination, the RNA samples are treated with RQ 1DNase-free RNase (Promega Corp., Madison, Wis.) and RT-PCR negativecontrols of RNased RNA samples were performed in parallel with theRT-PCR reaction. Each of the PCR reactions is ligated into the TAcloning vector pCR 2.1 (Invitrogen, San Diego, Calif.) and transformedinto frozen competent E. coli.

Nuclear Uptake of the Chimeric Molecules

Nuclear localization of fluorescently labeled chimeric oligonucleotideswas determined in the isolated cells. For in vivo studies, 250 l salinecontaining 75 g of fluorescently labeled chimeric oligonucleotidescomplexed to PEI is injected directly into the exposed caudate lobe. Theanimals are killed 24 h post injection, the tumor targeted is removed,bisected longitudinally, embedded using OCT and frozen cryosections werecut ˜10 pm thick, fixed, processed and examined using a MRC1000 confocalmicroscope (Bio-Rad, Inc., Hercules, Calif.).

In vivo Delivery of the Chimeric Oligonucleotides

Vehicle controls and lactosylated 25-kDa PEI at a ratio of 6 equivalentsof PEI nitrogen per chimeric phosphate are prepared in 300 l of 5%dextrose. The aliquots are administered either as a single dose of 100 gor divided doses of 150 g and 200 g on consecutive days. Five days postinjection, tumor tissue is removed for DNA and RNA isolation. DNA isisolated. RNA is isolated for RT-PCR amplification of the same region asthe genomic DNA using RNAexol and RNAmate (Intermountain ScientificCorp., Kaysville, Utah) according to the manufacturer's protocol.

Colony Hybridization and Sequencing

Eighteen to 20 h after plating, the colonies were lifted onto MSIMagnaGraph nylon filters (Micron Separations, Inc., Westboro, Mass.),replicated and processed for hybridization according to themanufacturer's recommendation. The filters were hybridized for 24 h with32P-end-labeled oligonucleotide probes (Life Technologies, Inc.,Gaithersburg, Md.), where the underlined nucleotide is the target ofmutagenesis. Hybridizations are performed at 37° C., and the filters areprocessed following hybridization for autoradiography. Plasmid DNAisolated from colonies identified as hybridizing with the 32P-labeledprobes is subjected to automatic sequencing using the forward andreverse primers, as well as gene specific primer corresponding tonucleotides of the normal gene.

EXAMPLE 2 Cells Transfected with Nucleic Acids Encoding SAgs

Cultured VX-2 carcinoma cells were shown to retain their tumorigenicactivity after implantation into New Zealand white rabbits. Progressivetumor outgrowth was observed over a 3 week period. Nucleic acid encodingSEB isolated and characterized by Gaskill et al, J. Biol. Chem. 263:6276(1988) and Ranelli et al., Proc. Natl Acad. Sci. U.S.A 82:5850 (1985)were used to transfect tissue cultured VX-2 carcinoma cells usingtransfection methodology described in Example 1. Transfectants wereselected using G418 and the survival of SEB-transfected VX-2 carcinomacells was observed. In additional experiments, attempts were made totransfect murine 205 and 207 tumor cells with nucleic acid encodingSEB(the kind gift from Dr. Saleem Khan) and Streptococcal pyrogenicexotoxin A (the kind gift of Dr. Joseph Ferretti). Successfiiltransfection of murine MCA 205 and B16 cells by nucleic acids encodingSEA and SEC2 was achieved shortly thereafter by integrating the SAg DNAinto several retroviral vectors (MFG NEO) containing a growth hormoneleader sequence under the control of a chick B-actin promoter (Krause JC et al., J. Hematotherapy 6: 41-51 (1997)). In addition, murine tumorsMCA 205 fibrosarcoma cells and a spontaneous mammary carcinoma cellswere successfully transfected with nucleic acids encoding SEB (providedby Dr. Saleem Khan) using the □-actin promoter. Transfected mammarycarcinoma cells induced T cell proliferation in vitro. To demonstratethe anti-tumor capacity of tumor cells transfected with nucleic acidencoding a SAg, these transfectants were injected i.p. into syngeneichosts with established mammary carcinomas. These transfectantsdemonstrated a capacity to reduce micrometastases of wild type mammarytumor in vivo assessed in a clonogenic lung metastases assay. Theanti-tumor effect produced by the SEB transfectants was enhancedsignificantly by the co-administration of tumor cells transfected withnucleic acids encoding the costimulating molecule B7-1.

EXAMPLE 3 Naked SAg DNA and Cells Co-Transfected with SAg DNA and withAdditional Nucleic Acid Encoding Anti-Tumor Motifs or Products

Nucleic acids encoding a SAg are injected in naked or plasmid form intoa host with cancer as a means of activating T cells and initiating ananti-tumor response. They may also be used as a vaccine to prevent theoccurrence or recurrence of tumor in a host. Under circumstances whereit is desirable to activate CD4 cells to produce a TH-1 cytokineresponse the nucleic acid construct used to transfect cells containsimmunostimulatory sequences such as unmethylated CpG sequences. Nucleicacids encoding SAgs may be co transfected into tumor cells together withnucleic acid encoding other constituents capable of promoting ananti-tumor response. A list of possible components of nucleic acidconstructs for direct administration and/or transfection of tumor cellswhich are administered to the host is presented in Table II.

The nucleic acid construct or constructs are administered to a hostintramuscularly, intradermally, systemically, parenterally,intratumorally, orally or locally (in the vicinity of the tumor).Alternatively, the construct is administered via a catheter or otherdevices known in the art into the tumor vasculature supplying all orpart of a tumor. When the construct is injected systemically, thenucleic acid construct is directed to the tumor using an anti-tumorantibody or ligand specific for a tumor receptor or receptor on thetumor neovasculature or stroma. The antibody or ligand or othertargeting structures are conjugated to the SAg nucleic acid construct inorder to facilitate the introduction of the construct into tumor cells.Nucleic acid/polypeptide complexes or nucleic acid/viral complexes areused to target a specific receptor on the tumor vasculature or stroma.TABLE II Nucleic Acid Constructs and Cells SAg-encoding DNA is usedalone or together with DNA encoding other cell surface moieties usefulin generating antitumor immunity. Genes or their products are shown incolumn 1, source information is shown in column 3, preferred cells to betransformed, transfected or transduced with the DNA are shown in column2. All of references are incorporated by reference in their entirety.Gene or Gene Product Cells transformed Reference or Source  1. SAg (SEQID NOS: 46-47) Tumor [See text]  2. Enterotoxin (SEQ ID NOS: 7-16) Tumor[See text]  3. SAg receptor (SEQ ID NOS: 46-47) Tumor [See text]  4.Enterotoxin receptor (SEQ ID NOS: 7-16) Tumor [See text]  5. CD1receptor(s) (SEQ ID NOS: 48-49) Tumor Martin LH et al., ProcNatl. Acad.Sci. 83: 9154-9158 (1986)  6. CD14 receptor (SEQ ID NOS: 50-51) TumorFerrero, E et al., J. Immunol. 145: 331-336 (1990)  7. CD44 encodingnucleic acids (SEQ ID NO: 52) T or NKT Nottenburg, C et al. Proc. Natl.Acad. Sci. 86: 8521-8525 (1989)  8. Carbohydrate modifying enzymes(SEQID NO: 53) Tumor, T or NKT Sheng, Y et al. Int. J. Cancer 73: 850-858(1997)  9. TCR Vβ&chain (SEQ ID NOS: 54-55 Tumor Tillinghast, JP et al.,Science 233: 879-883 (1986) 10. Staph/Strep hyaluronidase (SEQ ID NOS:57-58) Tumor Hynes WL et al., Infect. Immun., 63: 3015-3020 (1995) 11.Staph/Strep erythrogenic toxin (SEQ ID NOS: 58-59) Tumor McShan WM, etal., Adv. Exp. Med. Biol. 418: 971-973 (1997) 12. Staphylococcal□-hemolysin (SEQ ID NOS: 60-261) Tumor Projan SJ et al., Nucleic AcidRes. 3305-3309 (1989) 13. Strep capsular polysaccharide (SEQ ID NOS:62-63) Tumor Lin, WS et al., J. Bacteriol. 176: 7005-7016 (1994) 14.Staph staphylocoagulase (SEQ ID NOS: 64-65) Tumor Kaida S. et al., J.Biochemistry 102: 1177-1186 (1987) 15. Staph Protein A (SEQ ID NOS:66-67) Tumor Shuttleworth, HL et al., Gene 58: 283-295 (1987) 16. StaphProtein A domain D (SEQ ID NOS: 68-69) Tumor Roben, PW et al., J.Immunol. 154: 6347-6445 (1995) 17. Staph Protein A Domain B (SEQ ID NO:70) Tumor Gouda, H et al., Biochemistry, 31: 9665-9672 (1992) 18.Immunostimulatory protein Tumor, T or NKT Tokunaga, T et al., Microbiol.Immunol. 36: 55-66, (1992) 19. Costimulatory protein Tumor Entage, PC etal., J. Immunol. 160: 2531-2538 (1998) 20. SAg-mimicking nucleic acid Tor NKT 21. Glycophorin (SEQ ID NOS: 71-72) Tumor Siebert, PD. et al.,Proc. Natl. Acad. Sci. USA 83 1665-1669 (1986) 22. Mannose receptor (SEQID NOS: 73-74) Tumor Kim SJ. et al., Genomics 14: 721-727 (1992) 23.Angiostatin (SEQ ID NO: 75) Tumor Cao, Y. et al., J. Clin. Invest 101:1055-1063 (1998) 24. Chemoattractant (SEQ ID NOS: 76-77) Tumor Ames, RS.et al., J. Biol. Chem. 271: 20231-20234 (1996) 25. Chemokine (SEQ IDNOS: 78-79) Tumor Nagira, M et al., J. Biol. Chem. 272: 19518-19524(1997) 26. Transcription factor (SEQ ID NO: 80) Tumor, T or NKT Schwab Met al., Mol. Cell Biol. 6: 2752-2758 (1986) 27. Transcriptionfactor-binding Tumor, T or NKT    nucleic acid 28. SAg/peptide conjugateTumor 29. Glyco-SAg Tumor 30. Staph. global regulator gene agr (SEQ IDNOS: 81-83) Tumor Balaban, N. et al., Proc. Natl. Acad. Sci. USA 92:1619-1623 (1995) 31. Lipid A biosynthetic (SEQ ID NOS: 84-91) TumorSchnaitman CA et al.,    genes lpxA-D Microbiological Reviews 57:655-682 (1993) 32. Mycobacterial mycolic acid (SEQ ID NOS: 92-93) TumorFernandes ND et al., Gene 170: 95-99 (1996); Mathur M et al., J. Biol.Chem. 267: 19388-19395 (1992) 33. c-abl oncogene amplified in (SEQ IDNOS: 94-95) Tumor Scherle PA et al.,    chronic myel. Leukemia Proc.Natl. Acad. Sci. USA 87: 1908 (1990); Heisterkamp N et. al., Nature 344:251-253 (1990) 34. erbB2 (HER2/neu) oncogene (SEQ ID NOS: 96-97) TumorSchechter AL et al., Science 229: 976 (1985); Bargmann CL Nature 319: 22(1986); Hung MC et al., Proc. Natl. Acad Sci. 83: 261 (1986); Yamamoto Tet al., Nature 319: 230 (1986) 35. IGF-1 receptor gene (SEQ ID NOS:98-99) Tumor Abbott AM et al., J. Biol. Chem. 267: 10759-10763 (1992);Scott J et al., Nature 317: 260-262 (1985); Liu J et al., Cell 75: 59-63(1993) 36. VEGF (SEQ ID NOS: 100-101) Tumor Tischer E et al., J. Biol.Chem. 266: 11947-11954 (1991) 37. Strep emm-like gene family Tumor KehoeMA, In: Cell- Wall Associated Proteins in Gram-Positive Bacteria inBacterial Cell Wall, Ghuysen JM et al., eds, Elsevier, Amsterdam, 199438. iNOS(SEQ ID NOS: 102-103) Tumor Xie QW et al., Science 256: 225-228(1992) 39. Apolipoproteins (e.g., Lp(a), Tumor [See Text]    apoB-100,apoB-48, apoE) (SEQ ID NOS: 104-109) 40. LDL & oxyLDL receptors Tumor[See Text]    (e.g., LDL oxyLDL, acetyl-LDL,    VLDL, LRP, CD36, SREC,LOX-1,    macrophage scavenger receptors) (SEQ ID NOS: 110-121)Chemical Conjugation of SAg Nucleic Acids to VTs, Apolipoproteins, HPVEpitopes or Other Polypeptides/Proteins Listed in Tables I and II.

The following section describes actual physical conjugates between poly-or oligonucleotides and peptides or proteins. SAg nucleic acid_conjugates are prepared by chemical modification of nucleic acids atspecific sites within individual nucleotides or within oligonucleotidessuch that a protein can be bound to a DNA or RNA polymer.

Derivatization may be accomplished through discrete sites on theavailable bases, sugars, or phosphate groups to create primary amines,sulfhydryls, carboxylates or phenolates. The chemical modification ofnucleic acids can encompass several strategies. The initialderivatization may be the addition of a spacer arm to a particularreactive group on the nucleotide structure. Such a spacer typicallycontains a terminal functional group, such as an amine, that can be usedto couple another molecule. The spacer may be used to react with across-linking agent, such as a heterobifunctional compound that canfacilitate the conjugation of a protein or another molecule to themodified nucleotide.

If enzymatic methods are used to incorporate a small spacer into anoligonucleotide, subsequent chemical conjugation steps still are neededto add the protein moiety. In some cases, if an oligonucleotide containsthe appropriate functional group, a protein may be directly coupledusing chemical methods. Many of the chemical derivatization methodsemployed in these strategies involve the use of an activation step thatproduces a reactive intermediary. The activated species then can be usedto couple a molecule containing a nucleophile, typically a primaryamine.

A preferred method is to amidate the 5′ PO₄ of the oligonucleotide withEDC and then couple cystanmine to the 5′ amidated oligonucleotide. EDCwill add an amide to the oligonucleotide to form a phosphoramidatelinkage. After the addition of cystamine the disulfide is reduced withan agent such as dithiothreitol (DTT) to produce a free 5′ sulfhydryl.The derivatized oligonucleotide is then coupled to a protein chain(e.g., a verotoxin A or B chain) that has been activated with aheterobifunctional cross-linker such as succinimidyl4(N-maleimidomethyl)cyclohexane 1-carboxylate (SMCC ) which reacts withthe amines on the protein which then react with the sulfhydryls on thederivatized oligonucleotide. N-succinimidyl S-actylthioacetate (SATA) isuseful for adding a free thiol or sulfhydryl group to a molecule lackingthis moiety. With this modification, “protected” sulfhydryl is formedwhich may be stored indefinitely in this protected state.

When needed, the acetyl group on the protected sulfhydryl is removed toreveal the sulfhydryl for conjugation to another molecule. Aheterobifunctional agent such as SMCC or N-Succinimidyl3-(2-pyridylthio)propionate (SPDP) may be directly added to the amidatedoligonucleotide phosphate group to produce a free sulfhydryl unit forreactivity with the protein or peptide.

Chemical Conjugation of Polypeptides/Proteins to SAg DNA viaCarbodiimide Reaction with the 5′-Phosphates (Phosphoramidate Formation)

The water-soluble carbodiimide EDC, rapidly reacts with a carboxylate orphosphate to form an active complex able to couple with a primaryamine-containing compound. The carbodiimide activates an alkyl phosphategroup to a highly reactive phosphodiester intermediate. Diamine spacermolecules or anine-containing peptides then may react with this activespecies to form a stable phosphoramidate bond. Alternatively,bis-hydrazide compounds may be coupled to DNA using this protocol toyield a terminal hydrazide functional group able to react withaldehyde-containing molecules (Ghosh et. al., 1989). These methodspermit specific labeling of SAg DNA only at the 5′ end.

The following protocol describes the modification of SAg DNA or RNAoligonucleotides at their 5′-phosphate ends with a bis-hydrazidecompound, such as adipic acid dihydrazide or carbohydrazide. A similarprocedure for coupling the diamine compound cystamine is describedbelow.

Protocol

-   1. Weigh out 1.25 mg of the carbodiimide    1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride (EDC)    into a microfuge tube.-   2. Add 7.5 μl of SAg RNA or DNA that has 5′ phosphate groups. The    concentration of the oligonucleotide should be 7.5-15 nmol or a    total of about 57-115.5 μg. Also immediately add 5 μl of 0.25 M    bis-hydrazide compound dissolved in 0.1 M imidazole, pH 6.-   3. Mix (e.g., by vortexing) and centrifuge in a microfuge for 5 min    at maximal rpm.-   4. Add an additional 20 μl of 0.1 M imidazole, pH 6. Mix and allow    to react for 30 mm at room temperature.-   5. Purify the hydrazide-labeled oligonucleotide by gel filtration on    Sephadex G-25 using 10 mM sodium phosphate, 0.15 M NaCl, 10 mM EDTA,    pH 7.2. The oligonucleotide now may be conjugated with an    aldehyde-containing molecule.    Sulfhydryl Modification of SAg DNA

Creating a sulfhydryl group on SAg DNA allows conjugation reactions tobe done with sulfhydryl-reactive heterobifunctional cross-linkersproviding increased control over the derivatization process. Proteinsare activated with a cross-linking agent containing an amine-reactiveand a sulfhydryl-reactive end, such as SPDP, leaving thesulfhydryl-reactive portion free to couple with the modified DNAmolecule. Having a sulfhydryl group on the SAg DNA directs the couplingreaction to discrete sites on the nucleotide strand, thus betterpreserving hybridization ability in the final conjugate. In addition,heterobifunctional cross-linkers of this type allow two- or three-stepconjugation procedures which result in better yield of the desiredconjugate than do homobifunctional reagents.

Cystamine Modification of 5′ Phosphate Groups on SuperantigenNucleotides Using EDC

SAg DNA or RNA is modified with cystamine at the 5′ phosphate groupsusing the carbodiimide reaction described above. In some procedures, thereaction is carried out in a two-step process by first forming areactive phosphorylimidazolide by EDC conjugation in an imidazolebuffer. Next, cystamine is reacted with the activated oligonucleotide,causing the inidazole to be replaced by the amine and creating aphosphoramidate linkage. Reduction of the cystamine-labeledoligonucleotide using a disulfide reducing agent releases2-mercaptoethylamine and creates a thiol group.

Protocol

-   1. Weigh out 1.25 mg of the carbodiimide    1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride (EDC)    into a microfuge tube.-   2. Add 7.5 μl of SAg RNA or DNA that has 5′ phosphate groups. The    concentration of the oligonucleotide should be 7.5-15 nmol or a    total of about 57-115.5 μg. Also immediately add 5 μl of 0.25 M    cystamine in 0.1 M imidazole, pH 6.-   3. Mix (e.g., by vortexing) and centrifuge in a microfuge for 5 min    at maximal rpm.-   4. Add an additional 20 μl of 0.1 M imidazole, pH 6. Mix and allow    to react for 30 mm at room temperature.-   5. For reduction of the cystamine disulfides, add 20 μl of 1 M DTT    and incubate at room temperature for 15 mm. This will release    2-mercaptoethylamine from the cystamine modification site and create    the free sulfhydryl on the 5′ terminus of the oligonucleotide.-   6. Purify the SH-labeled oligo by gel filtration on Sephadex G-25    using 10 mM sodium phosphate, 0.15 M NaCl, 10 mM EDTA, pH 7.2. The    oligonucleotide now may be used to conjugate with an activated    protein containing a sulfhydryl-reactive group.    SPDP Modification of Amines on Superantigen Nucleotides

SAg DNA that has been modified with an amine-terminal spacer arm may bethiolated to contain a sulfhydryl residue. Theoretically, anyamine-reactive thiolation reagent may be used to convert an amino groupon a SAg DNA molecule into a thiol. A preferred reagent both forcross-linking and for thiolation reactions is the heterobifunctionalreagent SPDP. The NHS ester end of SPDP reacts with primary amine groupsto produce stable amide bonds. The other end of the cross-linkercontains a thiol-reactive pyridyldisulfide group that also can bereduced with DTT to create a free sulfhydryl. The reaction of a5′-diamine-modified SAg DNA oligonucleotide with SPDP proceeds undermildly alkaline conditions (optimal pH 7-9) yields thepyridyldisulfide-activated intermediate. This derivative can be used tocouple directly with sulfhydryl-containing compounds, or it may beconverted into a free sulfhydryl for coupling to thiol-reactivecompounds. In an alternative approach, 2,2′-dipyridyldisulfide is usedto create reactive pyridyldisulfide groups on a reduced5′-cystamine-labeled SAg oligonucleotide. This derivative then can beused to couple with sulfhydryl-containing molecules, forming a disulfidebond. Reduction of the pyridyldisulfide end after SPDP modificationreleases the pyridine-2-thione leaving group and generates a terminal-SHgroup.

Protocol

-   1. Dissolve the amine-modified SAg oligonucleotide to be thiolated    in 250 μl of 50 mM sodium phosphate, pH 7.5.-   2. Dissolve SPDP at a concentration of 6.2 mg/ml in DMSO to make a    20 mM stock solution. Alternatively, LC-SPDP may be used and    dissolved at a concentration of 8.5 mg/ml in DMSO (also makes a 20    mM solution). If the water-soluble Sulfo-LC-SPDP is used, a stock    solution in water may be prepared just prior to addition of an    aliquot to the thiolation reaction. In this case, prepare a 10 mM    solution of Sulfo-LC-SPDP by dissolving 5.2 mg/ml in water. Since an    aqueous solution of the cross-linker will degrade by hydrolysis of    the sulfo-NHS ester, it should be used quickly.-   3. Add 50 μl of the SPDP (or LC-SPDP) solution to the SAg    oligonucleotide solution. Add 100 μl of the Sulfo-LC-SPDP solution,    if the water-soluble cross-linker is used. Mix.-   4. Allow to react for 1 h at room temperature.-   5. Remove excess reagents from the modified SAg oligonucleotide by    gel filtration. The modified oligonucleotide now may be used to    conjugate with a sulfhydryl-containing molecule, or it may be    reduced to create a thiol for conjugation with sulfhydryl-reactive    molecules.-   6. To release the pyridine-2-thione leaving group and form the free    sulfhydryl, add 20 μl of 1M DTT and incubate at room temperature for    15 mm. If present in sufficient quantity, the release of    pyridine-2-thione is followed by its characteristic absorbance at    343 nm (ε=8.08×10³ M⁻¹ cm⁻¹). For many oligonucleotide modification    applications, however, the leaving group will be present in too low    a concentration to be detectable.-   7. Purify the thiolated oligonucleotide from excess DTT by dialysis    or gel filtration using 50 mM sodium phosphate, 1 mM EDTA, pH 7.2.    The modified oligonucleotide should be used immediately in a    conjugation reaction to prevent sulfhydryl oxidation and formation    of disulfide cross-links.    N-succinimidyl S-actylthioacetate (SATA ) Modification of Amines on    Superantigen DNA Nucleotides

SAg oligonucleotides containing amine groups introduced by enzymatic orchemical means may be modified with SATA to produce protected sulfhydrylderivatives. The NHS (N-hydroxylsuccinimide) ester end of SATA reactswith a primary amine to form a stable amide bond. After modification,the acetyl protecting group can be removed as needed by treatment withhydroxylamine under mildly alkaline conditions. The result is terminalsulfhydryl groups that can be used for subsequent labeling withthiol-reactive probes or activated-protein derivatives.

Protocol

-   1. Dissolve the amine-modified SAg oligonucleotide to be thiolated    in 250 μl of 50 mM sodium phosphate, pH 8.-   2. Dissolve SATA in DMF at a concentration of 8 mg/ml.-   3. Add 250 μl of the SATA solution to the oligo solution. Mix.-   4. React for 3 h at 37° C.-   5. Remove excess reagents by gel filtration.-   6. To deprotect the thioacetyl group, add 100 μl of 50 mM    hydroxylamine hydrochloride, 2.5 mM EDTA, pH 7.5, and react for 2 h.-   7. The sulfhydryl-containing oligonucleotide may be used immediately    to conjugate with a sulfhydryl-reactive label, or it can be purified    from excess hydroxylamine by gel filtration.    Conjugation of a Polypeptide to SAg DNA

As indicated, the DNA molecule must be modified to contain one or moresuitable reactive groups, such as nucleophiles like amines orsulfhydryls. The modifications that employ enzymatic or chemical methodscan result in random incorporation of modification sites or can bedirected exclusively to one end of the DNA molecule, e.g., 5′ phosphatecoupling.

Some of the more common procedures for preparing DNA-polypeptideconjugates are given below.

Polypeptide (e.g., VT) Conjugation to Cystamine-Modified SAg DNA UsingAmine- and Sulfhydryl-Reactive Heterobifunctional Cross-linkers

Cystamine groups are added to the 5′ phosphate of SAg DNA as describedabove. Once a sulffiydryl-modified DNA has been prepared, the followingprotocol may be used. The protein is activated with SPDP. Reacting theSAgic DNA probe in excess allows easy separation of uncoupled SAgoligonucleotide from conjugated molecules.

Protocol

-   1. Dissolve a 5′-sulfhydryl-modified SAg oligonucleotide in water or    10 mM EDTA at a concentration of 0.05-25 μg/μl. Calculate the total    nanomoles of oligonucleotide present based on its molecular weight.-   2. Add 0.15M NaCl, 10 mM EDTA, pH 7.2. Add the oligonucleotide    solution to the activated protein in a 10-fold molar excess.-   3. React at room temperature for 30 mm with gentle mixing.-   4. The protein-DNA conjugate is purified away from excess SAg    oligonucleotide by dialysis or gel filtration, or through the use of    centrifugal concentrators. Centricon-30 concentrators (Amicon) that    have a molecular weight cutoff of 30,000 are also used to remove    unreacted oligonucleotides. Since the polypeptide molecular weight    is approximately 140,000 and the conjugate is even higher, a    relatively small DNA oligomer will pass through the membranes of    these units while the conjugate will not. To purify the prepared    conjugate using Centricon-30s, add 2 ml of the phosphate buffer from    step 2 to one concentrator unit, then add the reaction mixture to    the buffer and mix. Centrifuge at 1000 g for 15 mm or until the    retentate volume is about 50 μl. Add another 2 ml of buffer and    centrifuge again until the retentate is 50 μl. Invert the    Centricon-30 unit and centrifuge to collect the retentate in the    collection tube provided by the manufacturer.    Administration of Peptide-DNA (pDNA), Naked DNA, or Protein or    Peptide Conjugates

Naked DNA, pDNA, nucleic acid-peptide or -polypeptide conjugates orgenetic fusion products are administered parenterally (for example, iv,ip, im, subcutaneously, intrathecally, intratumoral, rectally,transcutaneously) or orally. Administration may also be by a gene gunusing a 1 ml syringe and a 28 gauge needle. The nucleic acid isadministered intradermally or intramuscularly in a total volume of 100μl. A Tyne applicator is used to deliver doses of 1-1000 μg of DNA at 3×weekly intervals. SAg-encoding nucleic acid is injected directly intothe tumor. The nucleic acid either contains or does not containimmunostimulatory sequences that induce activation of T cells and skewthe response toward production of TH1 cytokines. For example, if nucleicacids encoding a tumor associated antigen are used then the nucleicacids are engineered to incorporate ISS sequences in order to fullyactivate a TH1 response. Likewise, if nucleic acid encoding a tumorassociated antigen is cotransfected with nucleic acid encoding a SAg,then one of the nucleic acid constructs is engineered to contain an ISS.

Viral DNA, nucleic acid expression cassettes or plasmids orbacteriophages encoding the constructs given in Table II may be used forin vivo immunization in place of naked DNA. Viruses may also acquire theαGal epitope after transfection into tumor cells which contain theα-galactosyltransferase enzyme either naturally or via transfection. Thevirus must possess the intact N-acetyllactosamine substrate for thegalactosyl-transferase in order to express the αGal. The virusesshedding from these cells will express the αCal epitope. The virus alsocontains peptide sequences for SAg and tumor associated antigen acquiredfrom the tumor cells which were previously transfected with nucleicacids encoding SAg and tumor antigen. The shed virus may also expressstaphylococcal or streptococcal hyaluronidase and capsularpolysaccharide sequences obtained from host tumor cell or accessorycells previously transfected with nucleic acids encoding these genes.The shed virus expressing Gal, SAg, hyaluronidase and capsularpolysaccharide is capable of initiating a potent tumoricidal responsewhen administered to hosts with established tumors or when used as atumor vaccine against potential tumors.

These constructs are also used as vaccines. Further, the nucleic acidconstruct is pre-processed ex vivo in muscle cells before selectivedelivery into host tumor tissue. Cationic liposomes or other liposomesor drug carriers well known in the art are used as vehicles to deliverthe nucleic acids in vivo.

The transfection process is also carried out ex vivo. Nucleic acidsencoding SAgs together with the nucleic acid constructs given in TableII are transfected into tumor cells of all types and antigen presentingcells such as MHC class I and class II as well as APCs expressing CD1and mannose receptors. These include but are not limited to DCs,immunocytes, monocytes, macrophages, and fibroblasts. SAg is transfectedalone or together with one or more of the above constructs given inTable II. The transfected cell expresses/secretes preferentially a SAgplus an immunogenic oncogene product, anti-angiogenesis factor,glycosylceramide, LPS or Gal. The transfectants present their geneproducts on cell surface receptors such as conventional MHC moleculesfor SAgs or in the case of the glycosylceramides or LPS on a CD-1 ormannose receptor. (APC). Glycosylated SAgs show preference forpresentation on mannose receptors.

EXAMPLE 4 SAgs, Tumor Antigens, Glycosylceramides, LPS's, Binary andTernary Complexes Applied to MHC Class I, Class II, CD1 or MannoseReceptors

CD1 represents a family of non-polymorphic antigen presenting moleculesunlinked to the MHC molecules expressed by most professional APCs. TheNKT cells that recognize CD1 presented antigens express NKR-P1, Ly49receptors, an invariant chain and a V8.2 variable region. With respectto these receptors, they share identity and their natural ligands withNK cells. Specifically, CD1 binds peptides with extended NH₂ and COOHtermini flanking the core binding motif. Long peptides (greater than 8to 10 amino acids) with amino acid residues at their hydrophobic bindingsites and greatly restricted anchors are preferred. This recognition ofCD1-presented antigens depends on the type and distribution of sugarresidues. Mycobacterial cell wall antigens namely mycolic acids andlipoarabinomannan also bind to CD1. Recently several glycosylceramides,in particular, monogalactosyl ceramides GalCer) were shown to bind toCD1 and to activate NKT cells Specifically, CD1 molecules are capable ofpresenting mannosides with 1,2 linkages and a phosphatidylinositol unit.CD1 bound antigens are recognized by NKT cells (/TCR positive; CD4 andCD8 negative). For instance, NKT cells are activated by alipoarabimannan (LAM) presented on CD1 receptors and become cytolyticwhile producing abundant INF.

In the present invention, a SAg bound to a monogalactosylceramide suchas GalCer is loaded onto CD1 or MHC class I or II receptors expressed byAPCs. The CD1 or MHC receptors are in soluble or immobilized formproduced by methods well described in the art. According to thisinvention, CD1 receptors present SAg polypeptides complexed with GalCerlipids or oligosaccharides to T cell and/or NKT cell population whichrecognize the conjugates and commence differentiation to tumor specificeffector cells. These ligands are be loaded on the CD1 receptorsequentially, simultaneously or as a preformed conjugates.Alternatively, they are positioned on the CD1 receptor after internalprocessing of their nucleic acid counterparts in the antigen presentingcells. These cells are then harvested and used for adoptiveimmunotherapy (Examples 7, 15, 16. 18-23). These complexes are alsouseful in vivo as a preventative or therapeutic antitumor vaccine(Example 14, 15, 16, 18-23).

SAgs and tumor associated antigen (TAA) are loaded sequentially on toclass II receptors of antigen presenting cells. Alternatively, preformedcomplexes of tumor associated antigen and SAg are loaded onto MHC classII receptors. The SAg may be in the native or glycosylated form. Thetumor associated antigen is also fused genetically to the □ chain of theMHC class II receptor. A SAg is added once the TAA is expressed bound tothe MHC class II. The sequence may also be reversed so that a SAg isgenetically processed and bound to the □ chain after which the TAA isadded. Consensus or repeating nucleic acid sequences shared by a tumorassociated antigen and a SAg are cloned into a single sequence andtransfected into APCs which display the consensus peptide in the contextof the class II receptor. Methodology for production of the fusion genesis well described in the art. (See Ausubel. F M et al., supra; Sambrook,J et al., supra) T cells or NKT cells are activated after exposure toSAg and TAA producing an expanded tumor specific T cell effectorpopulation which is useful in adoptive immunotherapy of cancer (Examples7, 15. 16, 18-23).

Antigen presenting cells in this system are chosen from a groupconsisting of DCs, fibroblasts, macrophages, and lymphocytes, but otherprofessional APCs or any other cell transfectants, phage displays orliposomes expressing the class I or class II receptors are also used.Alternatively, a tumor associated antigen is bound to an APC that ispharmacologically or genetically inhibited from antigen processing. SAgis added and the complex of SAg and protein bound to class II is thenpresented to a T cell population to produce a tumor specific effectorcell population for use in adoptive immunotherapy of cancer as inExample 15, 16, 18-23). These complexes are also useful in vivo as apreventative or therapeutic antitumor vaccine (Example 14, 15, 16,18-23).

Soluble SAg MHC class II proteins with covalently bound single peptidesare produced using a baculovirus system to express in insect cells twomurine class II molecules with peptides attached by a linker to the Nterminus of their □-chains (Kozono H. et al., Nature 369: 151-154(1994)). The resulting peptide is engaged by the peptide binding grooveof the secreted MHC molecule and this complex is recognized by T cellsbearing receptors specific for the combination. In this method, theapproximately 100 bp fragment encoding the SAg and a flexible linkerwith an embedded thrombin cleavage site is introduced in frame by thePCR just after the third codon of the b1 domain. This assures arecognizable leader peptide cleavage site and flexible link between theC-terminus of the foreign peptide bound in the cleft of the MHC moleculeand the N terminus of the b1 domain of SAg amino acids. Solublecomplexes consisting of receptors and various SAg are prepared in thisway and are used to activate T cells for use in adoptive immunotherapy.Similarly, preparations consisting of MHC class I receptors, CD1 ormannose receptors complexed with SAgs, glycosylceramides or LPS's areproduced which are useful in activating T cells or NKT cells foradoptive immunotherapy of cancer in protocols given in Examples 7, 15,16, 18-23). These complexes are also useful in vivo as a preventative ortherapeutic antitumor vaccine (Example 14, 15, 16, 18-23).

To produce complexes composed of SAgs with class I or II MHC or solubleDR α or □ (lacking the transmembrane domain) and TCR heterodimer, asoluble human TCR heterodimer which has specificity for various tumorassociated antigens bound to the human class I or II MHC molecules orhuman soluble CD1 molecules is used. A typical system for preparingternary SAg-tumor peptide-MHC or ternaryCD1-glycosylceramide (preferablyGalCer)-SAg complexes capable of triggering T cells or NKT cells is asfollows. CD1, DR-1 or HLA-A2 restricted tumor antigen specific T cell orNKT cell clones are used although primary unsensitized T or NKT cellsmay be used as well. The DR-1 and HLA-A2 homozygous Epstein-Barrvirus-transformed B cell line LG-2 or DCs expressing CD1 receptors areused as APCs either live or fixed in 0.5% paraformaldehyde for 20minutes. LG-2 and DCs (2.67×10⁵ per ml) in RPMI 1640 with 1% fetalbovine serum are pulsed with tumor antigen and glycosylceramiderespectively for 2 hours at 37° C. and then washed in RPMI 1640/1% fetalbovine serum to remove unbound antigen. SAg is added for 2 hours at 37°C. Pulsed APCs (4×10⁴ per well) are co cultured with resting T cells orNKT cells (2×10⁴ per well) in round-bottom microtiter plates in RPMI1640/5% human serum, Twenty four hours later, the cells are harvested.The APCs are separated and the T cells or NKT cells may be optionallyexpanded further with IL.-2 Optionally, complexes comprising solublerecombinant DRα or □ chain with bound superantigen are presented to theT cell or NKT cells which are then expanded with IL-2. These cells arethen harvested and used for adoptive immunotherapy (Examples 7, 15, 16.18-23). The APC containing the complexes are also useful in vivo as apreventative or therapeutic antitumor vaccine (Example 14, 15, 16,18-23).

Also useful for tumor therapy are the complexes LIP⁺ GPI-SAg (fromSection 38), either free or in the form of vesicles or exosomescomprising SAg-GalCer complexes or SAg-tumor peptide (including but notlimited to normal mutated structures). The ternary complexes ofSAg-GalCer-heat shock protein and tumor peptide-heat shock protein arealso useful, These complexes may be in or soluble or immobilized form,attached to a CD1 or MHC or as part of a vesicle or exosome. thecomplexes are also useful in vivo as a preventative or therapeuticantitumor vaccine (Example 14, 15, 16, 18-23).

The tumor associated antigen or SAg-tumor associated antigen complex isconjugated to oxidized mannan (polymannose) by methods described byApostolopoulos, V et al., Proc. Natl. Acad. Sci. USA 92: 10128-10132(1995) which is then loaded onto mannose receptors of antigen presentingcells for stimulation of a T cell anti-tumor response. Alternatively,the SAg (optionally conjugated to tumor peptides)-mannan conjugate isadministered to tumor bearing hosts by methods in Example 15, 16,18-23).

The SAg alone or conjugated to a tumor associated antigen is recognizedby the mannose receptor on macrophages. This requires a glycosylated SAgwhich is recognized by the mannose receptor on macrophages. A native orglycosylated tumor associated antigen-SAg conjugate or a consensuspeptide of both polypeptides is presented to mannose receptors expressedon antigen presenting cells which are exposed to a T cell or NKT cellpopulation to produce a tumor specific effector cells by methods inExample 15, 16, 18-23). These complexes are also useful in vivo as apreventative or therapeutic antitumor vaccine (Example 14, 15, 16,18-23). They are also used ex vivo to produce a population of tumorspecific effector T or NKT cells for the adoptive immunotherapy cancerby methods and protocols given in Examples 7, 15, 16, 18-23 and 36.

The mannose receptor delivers the complex to the late endosomal andlysosomal vesicles and the MHC class II loading compartment where theantigen is loaded onto CD1b molecules. The C1b molecule is endocytosedat the plasma membrane in coated pits and vesicle structures, transitsto early endosomes and is then delivered to the MHC class II antigenloading compartment. The endosomal localization motif on the tail of theCD1b molecule is essential for antigen trafficking of CD1b through thelysosomal compartment required for loading of antigen into CD1b and itsultimate transport to the membrane. The antigen binding groove of CD1 isdeeper and narrower than the MHC class I molecule groove containing ahydrophobic binding site which accommodates the lipid portion of themolecules such as lipoarabinomannan or GalCer and the SAg-LPS constructsgiven herein. APCs expressing the above constructs are exposed to NKTcell populations which recognize the antigens in the context of the CD1receptor. If carried out ex vivo this results in the formation of tumorspecific effector NKT cells which are used for adoptive immunotherapy byprotocols given in Examples 7, 15, 16, 18-23).

EXAMPLE 5 SAg Conjugation to Glycosylceramides Gangliosides LPS's,Glycans, Peptidoglycans Lipoproteins, oxyLDL and Lipoarabinomannans

Selection of the SAg peptide to be used for coupling is governed byseveral criteria. In practice, a 10-15 residue peptide is selected. ForSAgs, the sites chosen for coupling are those presumed not to be vitallyinvolved in T cell binding and activation. In most SAgs, these sites arebroadly distributed throughout the molecule. They are available atflexible regions of the protein and on reverse turns or loop structures.C termini are more mobile than the rest of the molecule and frequentlyexposed on the protein surface. This region is accessible to be coupledto another ligand especially usingm-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) via a Cys residuethat has been added to the N terminus of the peptide. By coupling thepeptide via its N-terminal end, the peptide is exposed in a fashionsimilar to that found in the native antigen. Additional criteria forselection of the coupling site such as exposed hydrophilic regions,secondary structure, hydropathicity profiles, and probability of helixformation may not be useful. However, care is take not to disruptpredicted polysaccharide attachment sites, most notably the sequenceAsn-X-Ser or Asn-X-Thr, which predicts the presence of Asn-linkedpolysaccharide moieties. In addition to location of transmembraneregions, Asn-linked glycosylation sites and sites of signal sequencecleavage are all important. After due consideration, the C using 7-15residues terminus is preferred and is modified to accommodate MBS. Thisprocedure requires a free sulfhydryl group on the synthetic peptide andfree amino groups on the ligand. Therefore, to use this method, it isnecessary to add a Cys residue to the C or N terminus of the peptide.

Biochemical Conjugation Methods:

SAgs are conjugated to polysaccharide containing structures usingseveral methods well described in the art (Hermanson, GT BioconjugateTechniques Academic Press, San Diego, Calif. 1996). Two methods aregiven here one utilizing the isolated complex carbohydrate obtained fromthe purified ganglioside which is then chemically conjugated to SAg andin another method wherein the ganglioside and SAg are both incorporatedinto a liposomal membrane. Either method is used to produce complexeswhich are included within the scope of this invention. However they areby no means exhaustive of all the techniques which could be employed toconjugate human tumor antigens to SAg molecules. Other conjugationstrategies may be utilized to produce an immunologically active complexas described by this invention. (See Offord, R E. in Protein Engineeringed. A R Rees, Oxford, 1992)

Direct Conjugation of Ganglioside, LPS or Peptidoglycan to SAg Molecules

-   -   1. Ganglioside or LPS antigens are purified and are then        dissolved in aqueous solution at pH 6.0 at a concentration of        1.0 mM/ml    -   2. Endoglycoceramidase from Rhodococcus (Genzyme) is added to        the ganglioside solution to a level of 5 milliunits. The        solution is incubated overnight at 37° C. with gentle agitation.        The endoglycoceramidase specifically cleaves at the        ceramide-polysaccharide bond liberating ceramide and clipping        off the complex carbohydrate making up the ganglioside    -   3. The polysaccharide is isolated by HPLC size exclusion        chromatography or by ultrafiltration    -   4. SAg is dissolved in 1M sodium phosphate, 0.15 M NaCl, pH 7.5,        at a concentration of 1 mg/ml. The purified polysaccharide        antigen is added to this solution to a concentration of at least        1 mM/ml.    -   5. In a fume hood, 20 microliters of 5 M sodium cyanoborohydride        solution in 1 M NaOH (Aldrich) is added to each ml of the SAg        solution.    -   6. The reaction is mixed gently and incubated at room        temperature for 72 hours or 4° C. for 1 week. This reaction        reductively aminates the reducing end of the polysaccharide (at        the point it was cleaved by the endoglycoceramidase) to the        amine groups on the SAg protein creating stable conjugate        coupled through a secondary amine linkage. The degree of        polysaccharide coupling can be controlled by limiting the time        of reaction.    -   7. Remove unreacted carbohydrate and cyanoborohydride by gel        filtration on Sephadex G-25 or by dialysis.

In a second method, SAg-GalCer, SAg-GalCer-CD1, SAg-glycosphingolipid,or SAg-glycosphingolipid-CD1 complexes are produced which have the addedbenefit of presenting the glycosylceramide in a polyvalent array whichis important for high affinity binding to complementary receptors. Theyretain nearly all of their original structure including most of theceramide moiety and the entire oligosaccharide chain. The principle ofpreparation derived from Mahoney, J A et al., Meth. Enzymol 242: 17-27(1994) is as follows. The fatty acid amide is hydrolyzed from the intactganglioside converting it to its lyso form which has a unique primaryamine at the 2-position of sphingosine. The lysoganglioside is treatedwith a bifunctional cross-linking reagent, succinimidyl4(N-maleimidomethyl)cyclohexane 1-carboxylate (SMCC), which forms anamide bond to the 2-position of sphingosine and results in asulfhydryl-reactive maleimidyl moiety attached through a linker arm, tothe original position of the fatty acid amide on the ceramide portion ofthe ganglioside. The SAg protein is treated with a reagent,N-succinimidyl S-acetylthioacetate (SATA), which converts the lysinee-amino groups to acetylated sulfhydryls. Subsequent treatment withhydroxylamine reveals the desired free sulfhydryls. Treatment ofsulfhydryl-derivatized SAg with maleimidyl derivatized gangliosideresults in a stable thioester linkage between the ganglioside and theprotein. The final product is chromatographically purified andcharacterized by protein and carbohydrate analysis. The SAg-GalCer orSAg-glycosphingolipid complex is then loaded onto a soluble CD1receptor.

LPS's and peptidoglycans are conjugated to SAg by methods well describedin the art. The most convenient and preferred method to targetspecifically the polysaccharides on the protein is through mild sodiumperiodate oxidation. Periodate cleaves adjacent hydroxyl groups in sugarresidues to create highly reactive aldehyde functional groups. Thegenerated aldehydes are used to in coupling reactions with amine orhydrazide containing molecules to form covalent linkages. Amines reactwith formyl groups under reductive amination conditions using a suitablereducing agent such as sodium cyanoborohydride. The result of thereaction is a stable secondary amine linkage. Hydrazides spontaneouslyreact with aldehydes to form hydrazone linkages, although the additionof a reducing agent greatly increases the efficiency of the reaction andthe stability of the bond. (See Hermanson, G T. Bioconjugate Techniques,Academic Press, San Diego Calif. 1996).

Production of Liposomes Displaying Glycolipid or Apolipoprotein oroxyLDL-SAg Complexes

Liposomes composed of the highly immunogenic constructs described hereinare prepared. They may include lipoproteins such as SAgs coupled to Gal,GalCer or SAg-glycosphingolipid or and other glycosylceramides.Liposomes comprising SAgs conjugated to apolipoproteins or oxyLDLreceptors are useful for targeting endothelial or macrophage oxyLDLreceptors in tumor microvasculature. Cationic liposomes are also usefulas a means of transferring the nucleic acid constructs of this inventionto tumor tissue. GalCer (a monogalactosylceramide) comprises the majorportion of the liposome. The most effective lengths of fatty acyl chainand sphingosine (or ceramide) base are C₂₆ and C₁₈ respectively and aphytosphingosine backbone. Sphingolipids lend structural advantages tothe integrity of liposomal membranes and have prolonged duration invivo. The Gal carbohydrate epitope is linked to liposomes via theamphipathic properties of the surface sphingolipids. The Gal isconverted to a glycolipid with a sphingosine backbone possessing ahydrophobic fatty acid tail that embeds them into membrane bilayers. Thehydrophilic carbohydrate ends of these amphipathic molecules caninteract with molecules dissolved in the surrounding environment.Sphingosine glycolipids consisting of lactosylceramide,GalGal(1-3)Gal(1-4)GlcNAc-R) or glycosphingolipids with terminalGal(α1-4)Gal are prepared in a manner similar to that of sphingolipids.All methods of preparation of liposomes have several steps in common:(1) dissolution of the lipid mixture in an organic solvent, (2)dispersion in an aqueous phase, and (3) fractionation to isolate thecorrect liposomal population.

In the first stage, the desired mix of lipid components is dissolved inorganic solvent (usually chloroform:methanol (2:1 by volume) to create ahomogenous mixture. This mixture includes any phospholipid derivatizedto contain reactive groups as well as other lipids used to form andstabilize the bulk of the liposomal structure. The correct ratio oflipid constituents to form stable liposomes is important A reliableliposomal composition for encapsulating aqueous substances containsmolar ratios of lecithin:cholesterol:negatively charged phospholipid(e.g., phosphatidyl glycerol) of 0.9:1:0.1. Apolipoproteins (e.g.,LP(a)) or oxyLDL (e.g., 7□-hydroperoxycholesterol or7□-hydroperoxy-choles-5-en-3B-ol) can substitute for cholesterol in thepreparation of the liposomes. In general, to maintain membranestability, the PE derivative should not exceed a concentration ratio ofabout 1-10 mol PE per 100 mol of total lipid. Once the desired mixtureof lipid components is dissolved and homogenized in organic solvent,several techniques are used to disperse the liposomes in aqueoussolution. These methods are broadly classified as (1) mechanicaldispersion, (2) detergent-assisted solubilization, and (3)solvent-mediated dispersion. With mechanical dispersion to formvesicles, the lipid solution is dried to remove all traces of organicsolvent prior to dispersion in aqueous media. The dispersion process iskey to producing liposomal membranes of the correct morphology. Methodsutilized include simple shaking, high pressure emulsification,sonication, extrusion through small-pores membranes and variousfreeze-thaw techniques. Detergent-assisted solubilization is also usedto bring the lipid more effectively into the aqueous phase fordispersion. Triton X, alkyl glycosides or bile salts such as sodiumdeoxycholate are employed. Other modalities or dispersion include thesteps of dissolving phospholipids and other lipid to be part of theliposomal membrane in ethanol. This ethanolic solution is then rapidlyinjected into an aqueous solution of 0.16 M KCl using a Hamilton syringeresulting in a maximum concentration of no more than 7.5% ethanol. Usingthis method, single bilayer liposomes of about 25-nm diameter areproduced. To remove the excess aqueous components that are notencapsulated during the vesicle formation, gel filtration using SephadexG-50 or dialysis is employed. To fractionate the liposome populationaccording to size, gel filtration is carried out using a column ofSepharose 2B or 4B

SAgs are conjugated to the GalCer or glycosphingolipids with terminalGal(α1-4)Gal, apolipoproteins, LDL or oxyLDL or LDL receptors beforeincorporation into the liposomal membrane or they may be incorporatedinto the membrane during the preparation of the liposomal membrane.Likewise, the SAg is conjugated to GalCer or glycosphingolipids withterminal Gal(α1-4)Gal at the glycolipid's polar head region by methodswell known in the art including using heterobifunctional crosslinkers orperiodate oxidation techniques. Alternatively, after the GalCer orglycosphingolipids with terminal Gal(α1-4)Gal is incorporated into themembrane, the liposomes are derivatized for further binding to the SAgproteins using the sodium periodate which oxidizes the ceramide's freehydroxyl to an aldehyde which is further modified by reductiveamination. Using the phosphatidylethanolamine of the lipid in theliposome, SAgs are coupled to the liposome using various bifunctionalagents including carbodiimide, glutaraldehyde, dimethyl suberimidate,periodate oxidation followed by reductive, amination, N-succinimidyl3-(2-pyridyldithio)propionate (SPDP),succinmidyl-4-(p-maleimidophenyl)butyrate (SMPB), iodoacetate,succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC).

Two general methods are used to prepare immunogenic (i) SAg-GalCer, (ii)GalCerGal, (iii) GalCerGal-SAg and (iv) SAg-glycosphingolipid complexes:The molecules (1) are dissolved in solution and encapsulated within thevesicle construction, or (2) covalently coupled to phospholipidconstituents in the lipids using standard cross-linking chemicalreactions. Covalent coupling of SAg to liposomes is done through thehead groups using various phospholipid derivatives and cross-linkingchemical reactions. These are done via the PE molecules. Simpleencapsulation is also a viable technique as described in Hermanson(supra).

A sample method using periodate oxidation and reductive amination isgiven below.

-   1. A 5 mg/ml liposome suspension is prepared in 20 mM sodium    phosphate 0.15 M NaCl, pH 7.4. containing, on a molar ratio basis as    mixture of phosphatidyl choline:cholesterol:phosphatidyl glycerol of    8:10:1. Other liposome compositions may be used, for example methods    without cholesterol, as long as a periodate-oxidizable component    containing vicinal hydroxyls (e.g., phosphatidyl glycerol) is    present. Any method of liposome formation may be used that is common    to those skilled in the art including mechanical dispersion.-   2. Sodium periodate is dissolved to a concentration of 0.6 M by    adding 128 mg/ml of water. 200 ml of this stock periodate solution    is added to each mol of the liposome suspension with stirring.-   3. React for 30 min. at room temperature in the dark.-   4. The oxidized liposomes are dialyzed against 20 mM sodium borate,    0.15 M NaCl, pH 8.4, to remove unreacted periodate. This buffer is    ideal for the subsequent coupling reaction. Chromatographic    purification using a column of Sephadex G50 is also done. The    periodate-oxidized liposomes are used immediately to couple with SAg    molecules or they may be stored in a lyophilized state in the    presence of sorbitol for later use.-   5. SAg is added to the periodate oxidized liposome solution to    obtain a 1 mg/ml concentration.-   6. In a fume hood, add 20 ml of 5 M sodium cyanoborohydride solution    in 1 M NaOH (Aldrich) to each ml of the SAg solution.-   7. The reaction is mixed gently and incubated at room temperature    for 6 hours.-   8. Excess SAg and cyanoborohydride are removed by size exclusion    chromatography on a column of Sephadex G-50 or by dialysis using a    membrane with a molecular weight cutoff of 100,000 daltons.-   9. Ganglioside antigens isolated by the method described previously    are incorporated into SAg-containing liposomes by detergent    dialysis. An amount of ganglioside is added representing twice the    amount of phosphatidyl glycerol (on a molar basis) originally added    to form the liposome (prior to periodate oxidation). To this    solution, concentrated sodium deoxycholate is added to obtain a    final concentration of 0.7% (w/w) and mixed thoroughly using a    Vortex mixer. Finally, the liposome suspension is dialyzed against    PBS, pH 7.5. A sample of the encapsulation technique is given in    Hermanson, supra.

An additional method for preparation of liposomes containing GalCer orglycosphingolipids with terminal Gal(α1-4)Gal is as follows: The donorliposomes consist of liver phosphatidylcholine, dicetyl phosphate,cholesterol, 3-(Man1-3Man-sn-1,2diacylglycerol) and galactosylceramide.These are mixed in various percentages to permit optimal expression ofthe galactosylceramide. Constituent lipids in chloroform-methanol aremixed and dried under a stream of nitrogen. Buffer consisting of 0.15MNaCl, 10 MM sodium Phosphate, pH 7.4, 1 mM dithiothreitol, 0.02% NaN₃ isadded to the dried lipids at a volume of 1 ml per 0.9 μmol of lipidphosphorus in the donor liposomes. After a 30-min incubation at 25° C.,the lipids are dispersed into the buffer by sonication with a Bransomsonifier for 30 min under nitrogen at 0° C. The liposome suspension isused the same day after centrifugation at 1500 g for 30 min to removeany undispersed lipid and titanium fragments released from thesonication probe.

Liposomes used for transfer of nucleic acid constructs given herein haveunique structures as described below. A cationic liposome composed ofdimyristyloxypropyl-3-dimethyl-hydroxyl ammonium (DMRIE) with DOPE hasallowed up to 100 fold higher concentrations of lipid and DNA to beadministered in vivo with minimal toxicity. Improved transfectiontechniques have been observed with the DMRIE/DOPE of two to seven fold.The prototype cationic lipid for gene transfer is DOTMA(N[1-(2,3-dioleyloxy)propyl]-N,N,N-tri-methylammonium chloride) which ismixed with a equimolar amount of DOPE (dioleoylphosphatidylethanolamine). The lipid DOTMA/DOPE comprise the cationicliposome known a Lipofectin. For human studies, two different cationicliposomes formulations are used. The first includesDC-cholesterol(3b[N-(N′N′-dimethylaminoethane)-carbamoyl] cholesterol)mixed with DOPE. DC-cholesterol/DOPE is low concentrations has proven toreduce toxicity to cells in vitro, is metabolized in vivo, and hasprovided successful gene transfer into malignant tumors in humans (SeeExample 17 for use in humans).

Genetic Fusion of SAgs to LPS's

N-linked glycosylation occurs exclusively in the ER, whereGlc₃Man₉GlcNAc₂ is added to Asn residues present in the sequence Asn XSer/Thr (X, any residue except Pro). To produce a glycosylation site ona SAg capable of binding a LPS, recombinant vaccinia virus expressingSAg is produced with Gln149 or Asn149 directed to the ER by appendage ofNH₂-terminal ER insertion, The SAg is directed to the secretory pathwayusing signal sequence from IFN. Recombinant vaccinia viruses(rVVs)expressing TAP and SAg nucleoprotein are used. The full length SAg genemodified by standard molecular genetic methods to encode glycosylationsites is inserted into the thymidine kinase locus of vaccinia viruses(VVs) by homologous recombination as described using the pSX11 plasmidto express foreign proteins under the control of the VV p7.5 early/latepromoter. SAg nucleoprotein is directed to the secretory pathway usingthe signal sequence from IFN□. The SAg coding sequences of all of therVVs are verified by sequencing PCR-amplified copies of full-length NPgenes isolated from the rVV. The resulting SAg-LPS or SAg-lipoproteincomplexes are used to immunize a population of T or NKT effector cellsfor use in the adoptive immunotherapy of cancer (Examples 2, 5, 7 15,16, 18-23). They may be preloaded onto CD1 or MHC Class I or IIreceptors on APCs as described below in the course of ex vivoimmunization. These complexes may also be used in vivo as a preventativeor therapeutic antitumor vaccine as in Example 14, 15, 16, 18-23).

Preparation of Fusion Proteins

Preferred fusion proteins comprise SAgs linked to other proteins orpeptides such as VTs or their A and B subunits, IFNα receptors, CD19peptides or carbohydrate recognition units which are designed to targetthe SAg to glycosphingolipid receptors on tumor cells or α_(v)□₃ ligandArg-Gly-Asp or α_(v)□₅ ligand Asn-Gly-Arg in vivo or in vitro. Thesefusion proteins induce apoptosis of the tumor cells. The fusion proteinsare produced by conventional methods in a variety of cells using avariety of vectors such as phage λ regulatory sequences. Techniques arewell established for producing fusion proteins that include the lacZprotein(□-galactosidase), trpE protein, glutathione-S-transferase, andthioredoxin. Expression in E. coli is most conventional but baculoviralexpression systems are also useful. Fusion proteins are produced inbacteria by placing a strong, regulated promoter and an efficientribosome-binding site upstream of the cloned gene. Exemplified below isa procedure using a representative lacZ vector. However, it should berecognized that other vectors well known in the art would be useful.Plasmids encoding the above proteins are prepared as previouslydescribed.

Construction of Expression Plasmids and Detection of Fusion Proteins

-   1. The appropriate pUR (or pEX or pMR100) vector is ligated in-frame    to cDNA fragments to be expressed as fusion partners using the above    plasmids to create an in-frame fusion. cDNA encoding the verotoxins    may be obtained from Dr. G. Lingwood, University of Toronto; murine    p31 Ii are from Dr. R. Germain, National Institutes of Health and J.    Miller, University of Chicago.-   2. Bacteria of the following strains are transformed: E. coli K12    71/18 or JM103 with pUR vectors, M5219 with pEX vectors or LG90 for    pMR100 vectors. The cells are plated on LB medium containing    ampicillin (100 μg/ml) and incubated overnight at 37° C. (or 30° C.    in the case of the pEX vector). MacConkey lactose indicator plates    should be used for pMR100.-   3. Individual colonies are tested for the presence of the desired    insert by plasmid minipreps.-   If most of the colonies can be assumed to contain a cDNA (because    directional cloning or a dephosphorylated vector was used in step    1), they can be screened for protein production in parallel (see    step 4b). If not, clones that contain a cDNA, as determined by    plasmid minipreps, can be screened for protein expression later.    cDNA inserts into a pMR100 plasmid can be detected readily as red    colonies on the MacConkey lactose indicator plates.-   4. Colonies are screened as follows for expression of the fusion    protein.    -   a. Grow small cultures from 5-10 colonies in LB medium        containing ampicillin (100 μg/ml). Incubate overnight at 37° C.        (or at 30° C. for pEX).    -   b. Inoculate 5 ml of LB medium containing ampicillin (100 μg/ml)        with 50 μl of each overnight culture. Incubate for 2 hours at        37° C. (or at 30° C. for pEX) with aeration. Remove 1 ml of        uninduced culture, place it in a microfuge tube, and process as        described in steps d and e. If screening for protein production        is being done in parallel, prepare plasmid minipreps from 1-ml        aliquots of the overnight cultures.    -   c. Induce each culture as follows: For pUR or pMR100 vectors,        add isopropylthio-□-D-galactoside (IPTG) to a final        concentration of 1 nM and continue incubation at 37° C. with        aeration. For pEX vectors, transfer the culture to 40° C. and        continue incubating with aeration.    -   d. At various time points during the incubation (i.e., 1, 2, 3,        and 4 hours), transfer 1 ml of each culture to a microfuge tube,        and centrifuge at 12,000 g for 1 minute at room temperature in a        microfuge. Remove the supernatant by aspiration. The kinetics of        induction varies with different proteins, so it is necessary to        determine the time at which the maximum amount of product is        produced.    -   e. Resuspend each pellet in 100 μl of 1×SDS gel-loading buffer,        heat to 100° C. for 3 minutes, and then centrifuge at 12,000 g        for 1 minute at room temperature. Load 15 μl of each suspension        on a 6% SDS polyacrylamide gel. Use suspensions of cells        containing the vector alone as a control. (For pEX and ORF        vectors, also use □-galactosidase as a control.) The fusion        protein should appear as a novel band migrating more slowly than        the intense □-galactosidase band in the control. It is not        uncommon for a protein the size of □-galactosidase to be present        along with the fusion protein.        Composition of 1×SDS gel-loading Buffer-   50 mM Tris Cl (pH 6.8)-   100 mM dithiothreitol (DTT)-   2% SDS (electrophoresis grade)-   0.1% bromophenol blue-   10% glycerol-   1×SDS gel-loading buffer lacking dithiothreitol can be stored at    room temperature. Dithiothreitol should then be added, just before    the buffer is used, from a 1 M stock.    Loading of SAg-LPS or SAg-Lipoprotein Conjugates onto CD1 or MHC    Receptors

For loading of SAg LPS or SAg-lipoprotein complexes onto CD1 receptors,recombinant soluble CD1-2M complexes in Drosophila melanogaster cellsare used to screen a random peptide phage display library(RPPDL). Theabsence of peptide-loading machinery in D. melanogaster cells results inthe expression of class 1 molecules that are properly folded andfunctionally competent but essentially devoid of bound peptide. Thisapproach has been shown to be useful in defining peptide binding motifsfor classical and nonclassical MHC Class I and Class II molecules.(Jackson et al., Proc. Natl. Acad. Sci. 89:1217-1224, 1992; Hammer etal., J. Exp. Med 175,1007-1012, 1992; Hammer et al., Cell 74, 197-201,1993). Each clone of SAg-lipoprotein contains a random 22-amino acidsequence at the mature NH₂ terminus of the gene VIII (filamentous coatprotein of the M13 bacteriophage). Recombinant soluble mCD1 isengineered with a C-terminal hemagglutinin (HA) tag, an epitope derivedfrom the influenza HA protein. In this way, the mCD1-phage complexes areidentified with a HA tag specific antibody. For immunizing usage,isolated receptor or antigen presenting cells of various types whichexpress CD1 or MHC class II molecule pretreated with formaldehyde may beused for loading the SAg-LPS or SAg-lipoprotein complexes. These APCswith bound complexes are then used to immunize T cells or NKT cells foruse in adoptive immunotherapy of cancer (Examples 2, 7, 15, 16 18-23).

Incorporation of Exogenous Lipid e.g. Glycolipid, Apolipoprotein oroxyLDL into Cells by Fusion with Liposomes

To prepare glycolipid, apolipoprotein, oxyLDL or Receptor containingliposomes, 400 μg of galabiosylceramide (Gb2) globotriosylceramide(Gb3), globotetraosylceramide (Gb4), galactosylceramide(GalCer),glucosylceramide (GlcCer) oxyLDL or apolipoprotein are driedwith 200 μg of phosphatidylethanolamine (PE) and 200 μg ofphosphatidylserine (PS) under a stream of nitrogen gas. 400 μl ofsterile isotonic PBS, pH 7.4, is added to the lipid, and the mixture issonicated using a water bath sonicator for 30 minutes. Liposomepreparations are used immediately.

To incorporate exogenous glycolipid into cells, tumor cells in latelogarithmic growth phase, sickled erythrocytes or vesicles (1.6×10⁷cells) are washed twice with PBS to remove serum proteins and thensuspended in serum-free RPMI 1640 medium at 4×10⁶ cells/ml. The cellsare incubated in the presence of the liposomes (or PBS for controls)prepared as above with rotary shaking (100 rpm) at 37° C. for 1 hr.,washed twice (5 min. 800×g) with PBS. and incubated for 18-24 hr at 37°C. in the presence of medium supplemented with 10% fetal calf serumprior to use.

EXAMPLE 6 Targeting SAg Nucleic Acids, Phage Display Systems andPolypeptides to Tumor Sites

Parenterally administered nucleic acid is targeted to a particular cellpopulation as follows. Nucleic acid is attached to a desialylatedgalactose moiety that targets asialo-orosomucoid receptors in livercells. Nucleic acid is attached to other ligands such as transferrin andTAP-1 as well as antibodies to surface structures such as the Le^(y)receptor. These ligands and antibodies bind to surface structures andare internalized. Thus, the attached nucleic acid is delivered to a cellof choice.

Sickled Erythrocytes as Gene Carriers

Erythrocytes from patients with sickle cell anemia contain a highpercentage of SS hemoglobin which under conditions of deoxygenationaggregate followed by the growth and alignment of fibers transformingthe cell into a classic sickle shape. Retardation of the transit time ofsickled erythrocytes results in vaso-occlusion. SS red blood cells havean adherent surface and attach more readily than normal cells tomonolayers of cultured tumor endothelial cells. Reticulocytes frompatients with SS disease have on their surface the integrin complex α₄□₁which binds to both fibronectin and VCAM-1, a molecule expressed on thesurface of tumor endothelial cells particularly after activation byinflammatory cytokines such as TNF, interleukins and lipid-mediatedagonists (prostacyclins). Activated tumor endothelial cells aretypically procoagulant. Similar molecules are upregulated on theneovasculature of tumors. In addition, upregulation of the adhesive andhemostatic properties of tumor endothelial cells are induced by viruses,such as herpes virus and Sendai virus. Sickled erythrocytes lackstructural malleability and aggregate in the small tortuousmicrovasculature and sinusoids of tumors. In addition, the relativehypoxemia of the interior of tumors induces aggregation of sicklederythrocytes in tumor microvasculature. Hence, sickled erythrocytes withtheir proclivity to aggregate and bind to the tumor endothelium areideal carriers of therapeutic genes to tumor cells.

Red blood cell mediated transfection is used to introduce variousnucleic acids into the sickled erythrocytes. The extremely plasticstructure of the erythrocyte and the ability to remove its cytoplasmiccontents and reseal the plasma membranes enable the entrapment ofdifferent macromolecules within the so-called hemoglobin free “ghost.”Combining these ghosts and a fusogen such as polyethylene glycol haspermitted the introduction of a variety of macromolecules into mammaliancells (Wiberg, F C et al., Nucleic Acid Res. 11: 7287-7289 (1983);Wiberg, F C et al., Mol. Cell. Biol. 6: 653-658 (1986); Wiberg, F C etal., Exp. Cell. Res. 173: 218-227 (1987). Both transient and stableexpression of introduced DNA are achieved by this method. Sickled cellscan also be transfected with a nucleic acid of choice e.g.,apolipoproteins, RGD in the nucleated prereticulocyte phase(e.g.proerythroblast or normoblast stage) by methods given in Example 1.Sickled erythrocytes transfected with nucleic acids encoding a SAgand/or carbohydrate modifying enzyme to induce expression of the a Galepitope, apolipoproteins, RGD and/or any construct described herein.Nucleic acids encoding additional polypeptides alone or together withSAg as described in Tables I and II to including but not limited toangiostatin, apolipoproteins, RGD, streptococcal or staphylococcalhyaluronidase, chemokines, chemoattractants and Staphylococcal protein Aare transfected into and expressed by sickled erythrocytes. These sicledcell transfectants are administered parenterally and localize to tumorneovascular endothelial sites where they induce a anti-tumor response.The methods of in vivo transfection of tumor cells are given in theExamples 17. Protocols for use of these transfectants in the inductionof anti-tumor immune response are described in Examples 14, 15, 16,18-23, 31

Vesicles from Sickled Erythrocytes

Vesicles from sickled erythrocytes are shed from the parent cells. Thecontain membrane phospholipids which are similar to the parent cells butare depleted of spectrin. They also demonstrate that a shortenedRussell's viper venom clotting time by 55% to 70% of control values andbecome more rigid under acid pH conditions. Rigid sickle cell vesiclesinduce hypercoagulability, are unable to pass through the spleniccirculation from which they are rapidly removed. Sickled erythrocytesare transfected in the nucleated prereticulocyte phase with superantigenand apolipoprotein nucleic acids as well as RGD nucleic acids. Nucleicacids encoding additional polypeptides alone or together with SAg asdescribed in Tables I and II are transfected into and expressed bysickled erythrocytes. Any of the the immature or mature sicklederythrocytes and their shed vesicles expressing the molecules given inTables I and II are capable of localizing to tumor microvascular siteswhere they bind to apolipoprotein receptors and induce an anti-tumoreffect. Because of their adhesive and hypercoagulable properties as wellas their rigid structure, these sickled cell vesicles expressingsuperantigen and apolipoproteins are especially useful for targeting thetumor microvascular endothelium and producing a prothrombotic,inflammatory anti tumor effect. Sickled erythrocytes and their vesiclesare capable of acquiring oxyLDL via fusion with oxyLDL containingliposomes as in Example 5. The resulting sickle cell or liposomeexpresses oxyLDL alone or together with SAg. Binding of oxyLDL to theSREC receptor on tumor microvascular endothelial cells induces apoptosisand simultaneous superantigen deposition produces a potent T cellanti-tumor effect.

Vesicles are prepared and isolated as follows: Blood is obtained frompatients with homozygous sickle cell anaemia. The PCV range is 20-30%,reticulocyte range is 8-27%, fetal hemoglobin range is 25-13% andendogenous level of ISCs is 2-8%. Blood is collected in heparin and thered cells are separated by centrifugation and washed three times with09% saline. Cells are incubated at 37° C. and 10% PCV in Krebs-Ringersolutions in which the normal bicarbonate buffer is replaced by 20 mMHepes-NaOH buffer and which contains either 1 mM CaCl2 or 1 mM EGTA. Allsolutions contain penicillin (200 u/mI) and streptomycin sulphate (100ug/mI). Control samples of normal erythrocytes are incubated in parallelwith the sickle cells. Incubations of 10 ml aliquots are conducted ineither 100% N2 or in room air for various periods in a shaking waterbath (100 oscillations per mm). N2 overlaying is obtained by allowingspecimens to equilibrate for 45 mm in a sealed glove box (Gallenkamp)which was flushed with 100% N2. Residual oxygen tension in the sealedbox was less than 1 mmHg. The percentage of irreversibly sickled cellsis determined by counting. 1000 cells after oxygenation in room air for30 mm and fixation in buffered saline (130 mM Cl, 20 mM sodiumphosphate, pH 74) containing 2% glutaraldehyde. Cells whose length isgreater than twice the width and which possessed one or more pointedextremities under oxygenated conditions are considered to beirreversibly sickled. After various periods of incubation, cells aresedimented at 500 g for 5 mm and microvesicles ) are isolated from thesupernatant solution by centrifugation at 15,000 g for 15 mm. Themicrovesicles form a firm bright red pellet sometimes overlain by apink, flocculent pellet of ghosts (in those cases where lysis wasevident) which is removed by aspiration. Quantitation of microvesiclesis achieved by resuspension of the red pellet in 1 ml of 05% Triton X100followed by measurement of the optical density of the clear solution at550 nm. Optical density measurements at 550 nm give results that arerelatively the same as measurements of phospholipid and cholesterolcontent in the microvesicles. Cell lysis is determined by measurement ofthe optical density at 550 nm of the clear supernatant solutionremaining after sedimentation of the microvesicles. Larger samples ofmicrovesicles for biochemical and morphological analysis are preparedfrom both sickle and normal cells following incubation of up to 100 mlof cell suspension at 37° C. for 24 h in the absence or presence ofCa²⁺. Ghosts are prepared from sickle cells after various periods ofincubation. The cells are lysed and the ghosts washed in 10 mM Tris HClbuffer, pH 73, containing 02 mM EGTA.

These vesicles are useful as a preventative or therapeutic vaccine as inExamples 15, 16, 18-23, 36.

Phage Displayed SAgs

Phages displaying or free tumor homing peptides ligands such as thetripeptides Arg-Gly-Asp and Asn-Gly-Arg which tripeptides bind to theintegrins α_(v)□₃ and α_(v)□₅, respectively, that are located on tumormicrovasculature, are conjugated to (1) a SAg peptide, (2) naked DNAencoding a SAg peptide or (3) phage displaying a SAg peptide. Theseconstructs are prepared as in Examples 3 and 5 and are further describedin Jackson R H. et al., In: Protein Engineering: A Practical Approach,A. R. Rees et al. (eds), pp. 277-301, Oxford Press, London, 1992.Similarly tumor cells or sickled cells transfected with and expressingSAgs and other molecules given in Tables I and II are also transfectedwith nucleic acids encoding RGD which facilitates their localization totumor microvasculature. These conjugates or transfectants areadministered i.v. and localize to the tripeptides' integrin receptorssituated on the tumor microvasculature. Neovascular endothelial cells towhich these constructs have been targeted are transfected bySAg-encoding DNA so that they express or secrete SAgs locally. Thisinduces potent local T cell activation and engender a tumoricidal immuneresponse. Protocols for use of such conjugates, i.e., (1) naked SAg DNAconjugated to the integrin-binding peptides or (2) naked SAg DNAconjugated to phage that display the integrin-binding peptides, andtransfectants in the induction of anti-tumor immune response aredescribed in Examples 7, 15, 16, 18-23, 31

Nucleic Acid and Nucleoprotein SAg Mimics

SAgs are often incapable of homing to tumor cells expressing SAgreceptors in vivo because of the existence of naturally occurringSAg-specific antibodies and the affinity of SAgs for class II receptorson a wide variety of cells. To solve this problem, DNA chromatography isused to identify oligonucleotides instead of SAg peptides that bind toSAg receptors which are naturally expressed on tumor cells. The SAgreceptor-specific oligonucleotides are conjugated to a SAg peptide witha functional TCR or NKT cell binding site. Oligonucleotides are alsosubstituted for peptides in the SAg molecule which bind to MHC classreceptors and naturally occurring SAg-specific antibodies. Theseconjugates are used to target SAgs to tumor cells in vivo that eitherendogenously express a SAg receptor or are pre-transfected with nucleicacid encoding a SAg receptor. These peptide-oligonucleotide complexesare prepared by chemical conjugation methods well known in the art. Suchreceptor specific oligonucleotides may have several fold greateraffinity for the SAg receptor compared to the native SAg. While thesepeptide-oligonucleotide complexes are used predominantly in vivo totarget tumor cells bearing SAg receptors, they are also used ex vivo tostimulate T cells to become tumor specific effector cell which areuseful for adoptive immunotherapy of cancer (Example 7, 15, 16, 18-23).

In appropriate recombinant bacteria, nucleic acids encoding the SAgreceptor binding site expressed on tumor cells are fused to nucleicacids encoding SAgs. The resultant SAg polypeptide construct consists ofthe amino acid sequence of a SAg and its SAg receptor binding site(which is overexpressed if desired). The SAg with its expressed oroverexpressed SAg binding site is useful in targeting tumor cellsexpressing SAg receptors after administration to a tumor-bearing host.In a related construct, the nucleic acid encoding a SAg with anoverexpressed SAg receptor specific binding site is fused to the nucleicacid encoding a native or chimeric SAg with its binding site fornaturally occurring antibodies and its MHC class II binding siteremoved, mutated or replaced by peptides from another SAg against whichthere are no known naturally occurring antibodies. The TCR binding andactivating region of this molecule is conserved. This resulting SAgpolypeptide molecule binds to SAg receptors on tumor cells but alsoretains its capacity to activate the TCR. It is administeredparenterally or orally to a tumor bearing host (orally to a coloncarcinoma patient) and will effectively target tumor cells with SAgreceptors (such as colon carcinoma cells) without being diverted bynaturally occurring antibodies or class II receptor bearing cellspresent in whole blood. As such, this construct is useful in producingan anti-tumor effect when administered to a tumor bearing host as inExample 18-23).

Using DNA chromatography techniques, nucleic acid specific for SAgreceptors on tumor cells are identified. These nucleotides areconjugated to SAg polypeptides which are optionally devoid of class IIbinding sites and naturally occurring antibody binding sites but withconserved TCR binding and activating sites. These constructs are usefulin targeting tumor cells bearing SAg receptors in vivo while retainingSAg amino acid sequences specific for the TCR which are capable ofproducing a tumor specific T cell population effective in adoptiveimmunotherapy of cancer. The selected amino acid sequences are deleted,replaced or added to the SAg molecules using molecular cloning and sitedirected mutagenesis techniques well established in the art.

EXAMPLE 7 General Ex Vivo Immunization Methods to Produce Tumor SpecificEffector Cells for Adoptive Immunotherapy of Cancer

Several days (3 to 60 days) after intratumoral immunization with anucleic acid construct described herein, tumor draining lymph nodes areremoved and placed in tissue culture. These cells are further expandedin vitro with SAg polypeptide for 2-4 days and/or IL-2 in vitro for atotal of 3-15 days. These T cells are then harvested and reinfused intothe host. T effector cells produced after in vivo immunization withnucleic acid encoding a SAg are expected to display potent anti-tumoractivity.

Cells transfected ex vivo, are administered to the host wherein theyactivate lymphocytes in a number of ways. In one embodiment, the initialstep involves in vivo immunization of hosts using various transfectantsand constructs as described in Table II. The transfected cells areintroduced into the host tumor, a nearby region, subcutaneously in closeproximity to regional lymph nodes, or the lymph nodes draining thetumor. Transfected cells types, constructs and agents used in this stepare given in Table II. Tumor cells are irradiated or treated withmitomycin C after transfection with nucleic acid encoding a SAg and/oranother polypeptide so that polypeptides are expressed and fixed on thecell surface and the tumor cells do not proliferate when administered tothe host. In another embodiment, the initial step involves in vivoimmunization of the tumor bearing host with transfectants, constructsand cells as described in Table III. These agents are administered inclose proximity to the regional lymph nodes with or without a bacterialadjuvant such as bacillus Calmette-Guerin (BCG) or Corynebacteriumparvum. The lymph node cells are harvested 10 days later and tissuecultured for further in vitro immunization/stimulation with SAg or SAgexpressing cells that, optionally, coexpress a tumor associated antigen,costimulatory molecule or antigen presenting molecule.

Cryopreserved autologous tumor cells for subsequent tumor vaccinationand culture are obtained from patients. Fresh resected tumors aredissociated under sterile conditions into single cell suspensions bymechanically mincing tumor into 5-mm3 pieces followed by enzymaticdigestion. Generally, 1 gm of tumor is digested in a minimum volume of40 ml of an enzyme mixture consisting of Hank's balanced salt solution(HBSS) containing 2.5 units/ml of hyaluronidase type V, 0.5 mg/ml ofcollagenase type IV, and 0.05 mg/ml of deoxyribonuclease type I (allcommercially available from Sigma Chemical Co.; St. Louis, Mo.). Thedigestion is performed at room temperature with constant stirring in atrypsinizing flask for 2 to 6 hours.

The resulting cell suspension is filtered through a layer of No. 100nylon mesh (Nytek: TETKO, Inc.; Briarcliff Manor, N.Y.) andcryopreserved in 90% human AB serum (GIBCO; Grand Island, N.Y.) plus 10%dimethyl sulfoxide (Sigma) at −178° C. in liquid nitrogen for subsequentimmunization and culture.

Tumor cells are used in native form, with dinitrophenyl (DNP) or otherhaptens conjugated to them and then irradiated or treated withcytostatic drugs prior to use. Optionally, the tumor cells aretransfected with nucleic acid encoding a SAg, and/or tumor associatedantigen, and/or antigen presenting molecule, and/or costimulatorymolecule, and/or adhesion molecule, and/or xenogeneic antigen, and/orcarbohydrate modifying enzyme. The nucleic acid is introduced by methodsgiven previously. The cells are then irradiated to a dose of 25 Gy ortreated or with cytostatic drugs, viable cells counted by trypan blueexclusion and the cells resuspended so that a volume of 0.2 to 0.4 mlcontains 1-2×10⁷ with or without ⁷ colony forming units of fresh frozenTICE BCG.

Patients are vaccinated intradermally (i.d.) at two sites approximately10 cm from superficial inguinal lymph nodes. If necessary, axillarylymph nodes are used. Lymph node regions with previous dissections orclinical evidence of tumor are avoided.

Accessory cells including DCs, fibroblasts, endothelial cells,monocytes, and macrophages are used after transfection with nucleic acidencoding a tumor associated antigen, and/or SAg, and/or xenogeneicantigen, and/or carbohydrate modifying enzyme. If desired, theseaccessory cells or APCs are transfected with recombinant viral vectorscontaining nucleic acid the encode a SAg, and/or tumor associatedantigen, and/or costimulatory molecule, and/or antigen presentingmolecule, and/or costimulatory molecule, and/or adhesion molecule,and/or xenogeneic antigen. These cells need not be irradiated prior toadministration. These cells are administered using the same cell numbersgiven above with or without BCG.

Alternatively, patients are vaccinated with various tumor associatedantigens and other agents as described in Table II. The agents are boundto MHC class I, class II or CD1 receptors or to cells expressing thesereceptors. They are also given alone in doses ranging from 0.1 to 10 mgemulsified in various adjuvants well described in the art. A vaccinationcourse includes up to 6 inoculations of the above agents at 1-3 weekintervals. TABLE III Single Step in vivo Immunization of Tumor BearingHosts with SAg Nucleic Acids Alone, Combined with Nucleic Acid EncodingOther Peptides and SAg Nucleic acids Conjugated to Polypeptides orLiposomes I. Intratumoral injection of nucleic acid 1. Direct injectionof SAg nucleic acids into tumor. 2. Direct i.v. or intra-arterialinjection of SAg nucleic acids into tumor microvasculature. a. SAgnucleic acids conjugated to a polypeptide ligand specific for a tumorcell, tumor stromal cell, tumor microvascular or neovascular cellreceptors b. Nucleic acid within liposomes containing a monoclonalantibody. 3. Recombinant viruses containing nucleic acid. a. Inactivatethe virus in the host with gancyclovir II. After in vivo immunization(3-14 days), harvest regional lymph nodes and place in tissue culture.III. Activate and expand lymphocytes. 1. Treat with SAg for 2 days. 2.Treat with IL-2 for 3 days. IV. Inject tumor specific effector T cellsinto host.

Regional lymph node cells draining tumor sites, lymphoid cells obtainedafter the above priming, peripheral blood T cells, and tumorinfiltrating lymphocytes (TILs) are suitable sources of T cells that areactivated to function as effector cells (T cells activated against thecancer cells). T cells are obtained from tumor infiltrating lymphocyteseither before or after tumor vaccine immunization in vivo by the methodsdescribed herein.

Approximately 10 days after in vivo immunization, an enlarged draininglymph node is removed and cultured. An immunized lymph node used hereinis exemplary. A single cell suspension of lymph node cells is obtainedby mechanical dissociation. Briefly, lymph nodes are minced into 2 mm³pieces in cold HBSS with a scalpel. The fragments are then pressedthrough a stainless steel mesh with a glass syringe plunger. Theresultant cell suspension is filtered through nylon mesh and washed inHBSS. Cultures are established in 300-ml culture bags (Livecell Flasks;Fenwal, Deerfield, Ill.) with 200 to 250 ml of culture medium (CM: RPMI1640 with 10% human AB serum, 2 mM fresh L-glutamine, 1 mM sodiumpyruvate, 100 mg/ml of streptomycin, and 50 mg/ml of gentamicin all fromGIBCO; Grand Island, N.Y.), containing 1-2×10⁵ lymph node cells/ml and1-4×10⁵ irradiated (60 Gy) tumor cells/ml. Optionally, the lymph nodecells are further separated into populations CD4+ CD8+ T cells, NKTcells and/+ T cells. Some SAg complexes are presented bound to MHC classII receptors and some such as SAg-LPS complexes or SAg-glycosylceramidecomplexes are presented bound to CD1 receptors either free or on APCcell surfaces.

After 24 hours, various SAgs or SAg transfected cell types (STCT) givenin Table III are added in doses of 10⁵ to 10 ⁷ cells for 8-72 hours. Thecells are harvested and used for in vivo administration at this point.Specific cell populations are selected such as those having a particularTCR V profile or expressing CD44 using magnetic beads or otherseparation techniques well known in the art. Optionally, the SAgactivated T cells are expanded. Recombinant IL-2 (Cetus, Emeryville,Calif.: provided by Cancer Treatment Evaluation Program, National CancerInstitute) is added at the initiation of the cultures at a concentrationof 600 IU/ml (1 Cetus unit=6 IU of IL-2). Culture bags are incubated at37° C. in humidified 5% CO₂. Cell counts from aliquots obtained fromrandom bags are followed to observe lymphoid cell proliferation. Lymphnode cells are harvested when cells reached maximal density, usuallyafter a total of 5-7 days in culture followed by IL-2 at 24 IU/ml for 3days. These intervals are shortened depending on the cell viability,CD44 expression, or V expression or other conditions that adverselyaffect survival, viability, or therapeutic success. TABLE IV Two Step invivo/in vitro Methods and Agents for Producing Tumor Specific Effector Tcells A. In vivo immunization with SAg transfected tumor cells,accessory cells, or virus. 1. Tumor cells transfected with: a. Nucleicacid encoding a SAg b. Nucleic acid encoding a tumor associated antigenc. Nucleic acid encoding a carbohydrate modifying enzyme 2. Accessorycells transfected with: a. Nucleic acid encoding a SAg b. Nucleic acidencoding a tumor associated antigen c. Nucleic acid encoding acarbohydrate modifying enzyme d. Nucleic acid encoding an MHC molecule3. Recombinant viruses containing: a. Nucleic acid encoding a SAg b.Nucleic acid encoding a tumor associated antigen c. Nucleic acidencoding a carbohydrate modifying enzyme d. Nucleic acid encoding an MHCmolecule B. *In vivo immunization with: 1. Irradiated tumor cells. 2.Tumor associated antigens. 3. Irradiated tumor cells conjugated withDNP. 4. Tumor associated antigen/SAg conjugate or fusion polypeptides.5. Naked nucleic or plasmid or phage displayed nucleic acid encoding aSAg or attached to liposomes or albumin microspheres. 6. Naked orplasmid or phage displayed nucleic acid encoding a SAg/tumor associatedantigen polypeptide conjugate. 7. Tumor cells or accessory cellstransfected with nucleic acids encoding structures given in Table IGroup IA, (pages 5 and 6) GM-CSF, IL-2 and other cytokines. (Berns, AJM.et al., Human Gene Therapy 6: 347-368 (1995). 8. Tumor cells transfectedwith nucleic acids encoding chemokines (T and NKT cell chemoattractants)and granulocyte chemoattractants (C3a, C5a, MAP). 9. SAg naked DNA fusedor in mixture with DNA or structures non-transfected given in Table 1 IAB and C (pages 5 and 6) C. Lymphoid cells from draining lymph nodes areharvested 3-21 days later and placed in tissue culture for furtherstimulation. They are divided into T cell, NKT cell and/T cellpopulations. Alternatively, T cells, NKT cells and/T cells are obtainedfrom the peripheral blood and also placed in tissue culture for furtherstimulation. D. In vitro stimulation of T or NKT cell populations toproduce tumor specific effector cells as described in “C” is carried outwith STCT (SAg transfected cell types) or with constructs alone orapplied to appropriate receptors on APCs. MHC class II APCs are used forpresentation of SAg constructs. APCs expressing mannose, or CD1 or CD14receptors are used for presentation of glycosylated SAg, SAg-LPScomplexes, SAg-peptidoglycan complexes or SAg-glycosylceramidecomplexes. Isolated MHC class I, class II, mannose, CD1 or CD14receptors immobilized on solid supports such as polystyrene plates maybe used in place of APCs methods well known in the art. In this formthey bind corresponding ligands in the constructs given above forpresentation to T cells or NKT cells. STCT include tumor cells,accessory cells, antigen presenting cells, prokaryotic cells,autologous, allogeneic or xenogeneic cells lines and viruses. Accessorycells include the following: DCs, monocytes, macrophages, endothelialcells, fibroblasts and NK cells. These cells are transfected withnucleic acids encoding SAgs in combination with the nucleic acids givenbelow. These nucleic acids may include the ISS sequence; SAg genes maybe used with or without the ISS sequence.*In vivo immunization may be by various routes, e.g.,, i.d., i.m., or asorganoids or in adjuvants proximate to regional lymph nodes e.g.,inguinal lymph nodes. For tumor peptide genes an ISS is useful as iscotransfection of MHC class I genes. For SAg and tumor associatedantigen genes, the ISS is useful.

Antibodies or Fab fragments having specificity for CTLA-4 are added withor without IL-2 at any point to expand the T cell population and avertapoptosis. The cells are washed once at the end of STCT incubation andbefore the addition of IL-2 and/or anti-CTLA-4 antibodies. TABLE V Exvivo Modes of Antigen Presentation to T Cells or NKT Cells to ProduceTumor Specific Effector Cells A. Tumor Cells, Accessory Cells, AccessoryCell/Tumor Cell Hybrids, e.g., DC/Tumor Cell) Transfected with: 1.SAg-encoding nucleic acid 2. SAg-encoding nucleic acid and tumorassociated antigen nucleic acids (to include arrays of tumor associatedepitopes) 3. SAg nucleic acid and MHC class I or II nucleic acids. 4.SAg-encoding nucleic acid and co-stimulatory nucleic acids. 5.SAg-encoding nucleic acid and adhesion molecule nucleic acids. 6.SAg-encoding nucleic acid and α-galactosyltransferase synthetic nucleicacids or xenogeneic species specific nucleic acids. 7. SAg-encodingnucleic acid and chemoattractant nucleic acids 8. SAg-encoding nucleicacid and glycosylceramide synthesis nucleic acids 9. SAg nucleic acidand lipopolysaccharide synthesis nucleic acids 10. SAg-encoding nucleicacid and microbial lipoprotein or polysaccharide or peptidoglycanmembrane or capsular synthesis nucleic acids 11. SAg-encoding nucleicacid and SAg receptor nucleic acids 12. SAg-encoding nucleic acid andCD1 receptor synthesis nucleic acids 13. SAg-encoding nucleic acid andCD14 receptor synthesis nucleic acids 14. SAg-encoding nucleic acid andSAg promoter and/or global regulator nucleic acids 15. SAg-encodingnucleic acid and oncogene and/or transcription factor nucleic acids 16.SAg-encoding nucleic acid and angiogenesis factor or receptor nucleicacids 17. SAg-encoding nucleic acid and growth factor receptor nucleicacids18. SAg- encoding nucleic acid and cell cycle protein nucleic acids19. SAg-encoding nucleic acid and heat shock protein nucleic acids 20.SAg-encoding nucleic acid and chemokine nucleic acids 21. SAg-encodingnucleic acid and cytokine nucleic acids 22. SAg-encoding nucleic acidand tumor suppressor nucleic acids 23. SAg-encoding nucleic acid andantigen processing and trafficking nucleic acids B. Additional in vitroStimulatory Agents (preferred receptor) 1. Tumor peptides (Class I orClass II) 2. Tumor peptide-SAg conjugates or fusion proteins (Class I orClass II). 3. Lipopolysaccharide-SAg conjugate (Class II or CD14) a.arabinose b. mycolic acid c. teichoic acid d. muramic acid(Staphylococcal cell wall glycoprotein) e. mannan proteoglycans f.chondroitin-sulfate 4. Glycosylated SAgs. (Class II or mannose) 5.SAg-glycosylceramide conjugates (class II or CD1) a. GalCer conjugate b.Gal conjugate 6. SAg-proteosome conjugates 7. SAg or glycosylated SAg orSAg-glycosylceramide conjugates or SAg- lipopolysaccharide orSAg-peptidoglycan conjugates coupled to proteosomes 8. SAg orglycosylated SAg or SAg-glycosylceramide conjugates or SAg-lipopolysaccharide conjugates or SAg-peptidoglycan conjugates expressedon or coupled to liposomes C. STCT or SAg-tumor peptide conjugates areincubated with in vivo immunized T cells or NKT cells for 2-4 days andthen with IL-2 for 2-5 days. D. The tumor specific effector cells arethen harvested and injected in doses of 10¹⁰-10¹² every 3-7 days for 1-6treatments. E. Viruses are transfected into tumor cells, accessorycells, antigen presenting cells, allogeneic or xenogeneic cells. Theyare pre-programmed with DNA for SAgs alone or in combination with genesgiven in D. They may also utilize the host genome to produce a new geneproduct as for example the host-galactosyltransferase. Viruses mayinclude the following: 1. Adenoviruses. 2. Vaccinia virus. 3. Equineencephalitis virus. 4. Influenza virus. F. In an additional method,tumor associated antigens are bound to MHC class I positive cells andused to activate T cells. SAg-lipopolysaccharide complexes andSAg-glycosylceramide complexes are bound to CD1 or class II receptors onAPCs. In addition, SAg-lipopolysaccharide complexes orSAg-glycosylceramide complexes are presented bound to class II positiveAPCs. Alternatively, unbound tumor associated antigen/SAg conjugates orfusion products are added at a 0.1 to 200 μg/ml dose for 2 days. This isfollowed by STCT incubation or by native or mutant SAg treatment for 2days.

For comparative analysis, peripheral blood lymphocytes (PBL) areobtained from patients the same day as the lymph node harvest. PBL areisolated by Ficoll-Hypaque gradients from 60 ml of heparinized bloodsamples. The PBL are placed in culture utilizing 24-well tissue cultureplates at the same cell density as lymph node cells. PBL are harvestedat maximal cell density and characterized by phenotype analysis andcytotoxicity.

T cells, NKT cells, and NK cells are isolated by well known methodsdescribed in the art (Colligan, J E et al., eds, Current Protocols inImmunology, John Wiley, New York, 1996).

PBL are separated by Ficoll/hypaque sedimentation. Cells are recoveredfrom the interface, washed in PBS, and pelleted. Peripheral bloodmononuclear cells enriched for MHC class I molecules or MHC class IImolecules are used to bind tumor associated antigens or tumor associatedantigen/SAg conjugates for in vitro or in vivo immunization.

Cryopreserved groups of autologous PBMCs are thawed, washed twice inPBS, resuspended at 5 to 8×10⁶ cells/ml in CM and pulsed with 1 mg/mlpeptide in 15 ml conical tubes (5 ml/tube) for 3 hours at 37° C. ThesePBMC stimulators are then irradiated at 3000 rads, washed once in PBS,and added to the responder cells at responder stimulator ratios rangingbetween 1:3 and 1:19.

Tumor infiltrating lymphocytes are isolated from fresh surgicalbiopsies. Briefly, tumor tissues are minced into 1-mm3 pieces that arethen dissociated into single cell suspensions in Dulbecco's modifiedminimum essential medium (Gibco; Grand Island, N.Y.) supplemented with10% heat-inactivated human AB serum (NABI, Miami, Fla.), 0.05%collagenase (type 4; Sigma Chemical Co., St. Louis, Mo.), and 0.002%DNase (type 1; Sigma) on a magnetic stirrer for 1 hour. Subsequently,the tissue digests are washed and passed through a nylon mesh and tumorinfiltrating lymphocytes and tumor cells are separated on discontinuous(75%/100%) Ficoll/Hypaque gradients.

Lymph node lymphocytes are obtained by mechanical dissociation oftissues, followed by washing in medium and centrifugation onFicoll/Hypaque gradient [Newell K A, et al., Proc. Natl. Acad. Sci. USA,88:1074 (1991)]. Cryopreserved suspensions of tumor cells/tumorinfiltrating lymphocytes are defrosted, washed, and separated byallowing tumor cells to adhere to the surface of plastic wells. Therecovered non-adherent tumor infiltrating lymphocytes are transferred to6-well plates and cultured in serum-free AJM-V medium (Gibco)supplemented with 6,000 U/ml of IL-2 (Cetus-Chiron, Emeryville, Calif.)for 8 days. Tumor cells are cultured as adherent monolayers inDulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10%(v/v) of fetal calf serum. Any activated lymphocytes can be used in themethod given above. In a preferred embodiment, lymphocytes expressing apredominant TCR V phenotype in tumor tissue or peripheral blood beforeor after treatment are isolated and expanded by standard procedures.

Antibodies to various TCR V□ subsets are immobilized on inert solidsupports and incubated with blood cells and/or tissue cells to includebone marrow and peripheral blood or lymphoid tissue cells and tumorinfiltrating lymphocytes. The bound T cells are eluted with variousbuffers. Suitable biocompatible inert supports include polystyrene,polyacrylamide, nylon, silica, and charcoal as well as others known inthe art. The supports are derivatized for covalent binding of antibodieswith agents well known in the art including heterobifunctionalcompounds, carbodiimide, and glutaraldehyde. The enriched population ofV-bearing T cells is then used for in vitro immunization with a SAg innative or mutant form capable of activating the dominant TCR V bearinglymphocytes. IL-2 is used to further expand the cell population asdescribed above.

Effector lymphocytes obtained after in vivo sensitization are stimulatedin vitro with tumor associated antigens bound to irradiated PBMC (whichact as stimulator cells) for 8-72 hours. DCs, macrophages, or otherclass I-bearing cells are used to present the tumor associated antigens.The T cells are then analyzed for TCR V and/or CD44 expression. An STCTexpressing a SAg is then added to the culture (1 picogram to 10microgram). If a given V predominance is noted after antigenstimulation, then an STCT or SAg known for its ability to specificallystimulate that V subset is selected for use in activation. Cultureproceeds for 18-72 hours. The TCR V and CD44 profile of stimulated Tcells are then rechecked. IL-2 (12-25 IU) and/or anti-CTLA-4 antibodiesare added for an additional 8-72 hours after which the cells areharvested for use. The optimal timing of STCT introduction after tumorantigen stimulation is between 3 and 14 days.

Antigen-presenting cells (APCs) of all kinds such as DCs, B cells ormacrophages with appropriate MHC class II molecule binding sites forsoluble SAgs are used or the SAgs are presented alone or in immobilizedform without APCs. Optionally, STCTs are used without APCs. Before IL-2administration, effector cells are re-stimulated weekly by washing andreplating in 24 well plates at a concentration of 2.5×10⁵ cells/ml inCM. This is continued for 3-10 cycles until enough cells are availablefor IL-2 expansion. T cells are cloned 7 days after the several cyclesof stimulation in 96-well round bottom plates at 0.3 cells/well with5×10⁴ stimulator tumor antigen-PBMC, SAg, or STCT and 25-50 Urecombinant IL-2 in a volume of 200 ml.

For long term growth, clones are transferred to 24 well plates and 1×10⁶cells/well and stimulated weekly with SAg or STCT plus optimally 5×10⁵tumor associated antigen-PBMC and 25-50 U/ml of IL-2. After clones growto greater than 2×10⁶ cells, the clones are maintained by culturing withSTCT only for 48 hours, washing to remove STCT, and replating in freshmedia for 5-7 days with 25-50 U/ml IL-2.

The initial incubation is with the selected tumor associated antigensuch as MART-1 for 1-3 days with the latter reagents followed by Vprofiling and re-stimulation with SAg by methods given above. The MART-1is presented attached to HLA-A1⁺ cells of PBMC. Cytotoxic activity istested after the first and/or second rounds of sequential stimulationwith tumor associated antigen and SAg given below.

The tumor-specific effector T cell population is immortalized as tumorspecific T cell hybridomas. These hybridomas are generated byimmunization in vitro of human T cells as described herein. The expandedT cells are then fused to a thymoma and cloned by limiting dilution orother methods well known in the art.

Cells are cultured in complete tumor medium composed of Eagle's minimalessential medium supplemented with 10 mM 2-mercaptoethanol, 10% fetalcalf serum, 10% Mishell-Dutton Nutrient cocktail, 100 U/ml penicillin G,and 200 mg/ml streptomycin sulfate. Other well known culture media canalso be used.

For SAg immunization in vitro, various antigen presenting cells are usedincluding MHC class II-positive T cells as well as those expressing CD1.Purified MHC class II or CD1 molecules alone or immobilized aresubstituted for APCs in some cases. Moreover, T cells are activated bysome SAgs without APCs when presented to T cells in immobilized form orin the presence of various cytokines such as IL-1, IL-2, IL-4, or IL-6or xenogeneic antigens. Various costimulants such as B7-1 and B7-2,adhesion molecules such as ICAM-1 and VCAM-1, or GalCer are usedtogether with SAgs and MHC class II positive APCs or immobilized MHCclass II peptides to augment the T cell or NKT cell response.

Tumor associated antigen immunization is also involved in the binding ofpeptides to MHC class I bearing APCs of multiple origins. Variouscytokines including, but not limited to, IL-1, IL-2, IL-4, IL-12, or LPSare used in vitro or in vivo to expand the antigen specific clone of Tcells and avert the development of T cell anergy.

Specialized Forms of Tumor Specific Effector Cells and Hybridomas

Tumor specific T or NKT cells with TCR V and/or CD44 selectivity areproduced by transfecting uncommitted stem cells with nucleic acidsencoding particular TCR V chains. Likewise, a T cell cloneoverexpressing CD44 is produced by transfecting T cells with nucleicacids encoding CD44. A hybridoma expressing a tumor associated antigenwith a dominant TCR V phenotype or CD44 expression is produced in thisway. Such a T cell hybridoma or cell line is stimulated exogenously by aSAg or a SAg mutant with a TCR V or CD44 selectivity corresponding tothat expressed predominantly by the T cell hybridoma. The result is aclone of tumor specific T cells capable of being expanded by exposure toSAg in vitro or in vivo.

CD44 expression is induced in a T cell, NKT cell or TCR/T cellpopulation after activation in vitro or in vivo with SAgs alone ortogether with any of the T or NKT cell stimulating constructs andmethods described herein. The in vivo and in vitro activation steps andimmunization protocols are given in Examples 7, 15, 16. 18-23. The CD44positive T cell population exhibits upregulated primary adhesionproperties and is capable of effectively trafficking and homing to tumorcells in vivo and particularly to sites of SAg (in native or nucleicacid form) injection i.e. tumor Nucleic acids encoding CD44 or acarbohydrate modifying agent will induce CD44 expression on the T cellsurface. A preferred in vivo method of use involves intratumoralinjection of SAg DNA into tumor sites which induces expression of CD44on T cells resulting in enhanced T cell trafficking to the site of SAgadministration.

T cells are genetically engineered to overexpress CD44 after SAgstimulation. This is accomplished by transfection of T cells or NKTcells with nucleic acid encoding CD44 as well as nucleic acids encodingglycosyltransferases. This results in the overexpression of CD44upregulation of the adhesive properties of CD44. Such CD44 enrichedclones are harvested after SAg stimulation, enriched, and administeredfor adoptive immunotherapy of cancer (Examples 6, 7, 15, 16, 18-23).

Additionally, T or NKT cell clones or hybridomas are produced whichexpress a chimeric TCR consisting of an invariant chain with specificityfor GalCer or and a chain that binds a SAg. The V region which isspecific for the SAg is overexpressed on the TCR permitting greaterresponsiveness to exogenous SAg. This chimeric TCR recognizes and isstimulated by an exogenous SAg with a TCR V selectivity corresponding tothe predominant TCR V phenotype of the T or NKT cell. Such T or NKT celllines are cloned and hybridomas produced by methods well known in theart.(Current Protocols in Immunology, pp. 7.21.-7.21.9 John Wiley, NewYork, 1991) The expanded clone of tumor specific T cells produced inthis way is useful for adoptive immunotherapy of cancer by methods givenin Examples 7. 15, 16, 18-23.

T cell clones are produced due to asynchronous TCR V locusrearrangements at low but significant frequency in which both TCR Vsegments are part of two functional TCRs. Such clones are produced fromuncommitted stem cells in which nucleic acid encoding two chains aretransfected, one having specificity for a tumor associated antigen andanother having SAg specificity. Hence, a clone of T cells with dual VTCR expression is produced which is capable of reacting with a tumorspecific and a SAg. This clone is expanded by binding either or bothligands. These expanded clones consisting of tumor specific effector Tcells are used for adoptive immunotherapy of cancer by protocols givenin Examples 7, 15, 16, 18-23).

T cells or NKT cells clones or hybridomas expressing TCR Vα and V□chains with specificity for GalCer and SAg, respectively, are producedby fusion of NKT cell DNA encoding the GalCer and SAg receptors with DNAfrom an appropriate thymoma. This GalCer receptor and SAg receptors areexpressed on the and chain of the TCR, respectively. Upon exposure toGalCer or SAg, these cells are further activated to express CD44 whichenhances their homing and adhesive properties. NKT or T cells expressinghigh levels of IFN, GM-CSF, and IL-10 are selected and cloned. The cloneof T cells producing IFN and expressing GalCer, SAg and CD44 is thenexpanded and immortalized. With its properties of tumor recognition, SAgand glycosylceramide activation, IFN production and effective in vivotrafficking, this T or NKT effector cell population is preferred foradoptive immunotherapy of cancer by methods given in Examples 7, 15, 16,18-23).

Additional measures to avert apoptosis and augment proliferationcapacity in SAg activated T cells include the use of anti-CD28antibodies and inhibition of CTLA-4 on T cells. CTLA-4 on T cells isblocked by specific antibodies or fragments. Alternatively, a T cellclone is used in which CTLA-4 is genetically deleted. When stimulated bySAg, these cells proliferate to a greater extent compared to SAg alone.Cell populations in which CTLA-4 is deleted or blocked are selected tohave a predominant V bearing lymphocyte population that is activatedafter in vivo or ex vivo tumor associated antigen stimulation. AfterCTLA-4 deletion or blockade, the appropriate SAg with V selectivity ischosen to expand this population. To avert uncontrolled proliferation invivo, the thymidine kinase gene of the HSV is co-transfected to enableelimination of these cells in vivo if desired.

Measures to produce an effector T cell population with an overexpressedTCR V and/or V chains specific for a given SAg involve the transfectionof nucleic acids encoding the desired V or V regions into T cells as inExample 1. To lower the activation threshold of the T cell or NKT cellsto SAg or SAg-tumor peptide-MHC or CD1, the T cell or NKT cells aretransfected with nucleic acid encoding a tyrosine kinase or other signaltransduction initiating molecules which can dimerize in the membranewith the TCR tyrosine kinases thereby lowering the threshold foractivating the signal transduction pathway. The deletion of the signaltransduction inhibitory region of the TCR to produce sustained signaltransduction is done by site directed mutagenesis as in Example 24.

EXAMPLE 8 Prevention of Anergy in T or NKT Tumor Specific Effector Cells

The SAg stimulated tumor specific effector T cells used for adoptiveimmunotherapy of cancer may not function when infused unless measuresare taken to prevent T cell anergy or activation-induced cell death(AICD) by interdicting the Fas mediated pathway. The Fas ligand (FasL)has been identified as a type II transmembrane polypeptide of the TNFfamily. These two related receptor-ligand systems signal apoptosisthrough closely related but distinct pathways. T cell phenotypes thathave diminished expression of Fas or FasL show delayed anergy inductionand shortened periods of non-reactivity compared to Fas-expressingcells. Activation-induced cell death (AICD) induced by SAgs in vitro orin vivo is averted using Fas-deficient T cells, including those withdown-regulated Fas or FasL receptors as well as those with masked orblocked Fas receptors. A Fas-IgG fusion protein is added during the SAgactivation phase to prevent AICD or anergy induction. Measures such asthose above (or by treating with anti-CTLA-4 antibodies or activation ofCD28 before, during, or after STCT stimulation) protect T cells fromanergy or AICD. In this way, these manipulations prolong T cell survivalin vitro and enhance tumoricidal activity in vivo after the T cells areactivated by tumor associated antigen plus SAg or tumor associatedantigen-SAg conjugates in vitro.

SAg nucleotide alone or fused to tumor peptide nucleotide may be furtherfused with an antisense nucleotide capable of inhibiting the apoptosispathway. When expressed in T cells, this combination of genes wouldpromote the generation of tumor specific effector T cells which would beresistant to AICD. Oligonucleotide antisense molecules that inhibit keysteps leading to apoptosis may be fused to SAg DNA in order to preventthe T cells from undergoing AICD. SAg DNA may also be fused with themulti-drug resistance (MDR) gene to make the T cells refractory tochemotherapeutic agents and sensitive to anti-apoptosis drugs. Certaindrugs or radiation may be used together with SAg DNA for additive orsynergistic inhibition of the apoptosis pathway in the doubly ormultiply transfected T cells.

SAg DNA may also be linked operatively to promoter genes such as thoseinducible by corticosteroids or heavy metals (e.g., the metallothioneinpromoter) and regulatory DNA sequences that act as T cell on/off sensorsresponsive to exogenous cytokines, inflammatory stimuli and changingexternal conditions such as oxygen tension and pH. A particularadvantage of SAg DNA is that its expression will promote V receptordownregulation and internalization so that these receptors areunavailable to exogenous SAg. SAg DNA is modified in several ways tointroduce protein binding sites for key transcriptional elements whichmay inhibit apoptosis. Insertion of such sites at the bending domains ofSAg oligonucleotides renders them capable of inducing key TH-1 cytokinesand cell proliferation while averting AICD. SAg DNA is also capable ofreversing the T cell anergy and signaling defect which may be localizedto the chain in cancer patients. This is accomplished by providingtranscriptional binding sites on the SAg DNA which bypass theconventional chain activating signals and the pathway to IFN and IL-2production. In the same way, SAg DNA also bypasses the defective signalby activating a complex that contains STAT-1 which binds a GAS-likepalindromic sequence located in the IFN response region of the FcRIgene. Such anergy in T cells may also be reversed by alternatecytoplasmic tails that are activated by SAg binding to the TCR V and Vchains. Moreover, nucleic acid encoding Protein A and especially domainD (that binds to the Ig VH₃ region) may be fused to SAg DNA in order tobring about activation of the IL-2 and IFN genes that resulting in Tcell proliferation and IFN production coupled with up-regulated surfacereceptors for the Ig VH₃ domain.

Anergy in SAg-activated tumor-specific T or NKT effector cells (orhybridomas) is known to be averted by in vitro or in vivoco-administration of IL-2, IL-1, LPS and tumor specific peptidesspecifically interfere with SAg driven anergy. Methods and doses for useof these agents with SAg activated T or NKT cells are given in Examples7, 15, 16, 18-23.

Tumor specific T or NKT effector cells or hybridomas prepared by variousmethods described above are administered according to the adoptivetherapy protocol of Examples 7, 15, 16, 18-23 (the preferred method).The experimental tumor models and human cancers for which theanti-cancer efficacy of these cells can be demonstrated are provided inExample 16.

EXAMPLE 9 Reactivation of Anergized Tumor-specific T or NKT Cells by SAgand SAg Receptors

Preferred tumor-specific effector cells for adoptive immunotherapy ofcancer are autologous T cells. However, in the course of tumor growth, Tcells become anergized to the host's own tumor and are incapable of anadequate immune response to the tumor. Dampened TCR-triggered responsesare caused by suppression of effector molecules that couple cell surfacereceptors to early and late intracellular signaling events. For example,basal and induced tyrosine phosphorylation of many signaling proteins isreduced due to deficits at multiple points, including the inositolphosphatase pathway. This down-regulates cytokine production anddecreases nuclear transcription factors of TH1 helper cells. Twofunctionally distinct signal transduction pathways are coupled to theTCR. Native or mutant SAgs activate anergic T cells via an alternatepathway without the conventional increases in Ca++ mobilization ordetectable phosphatidylinositol hydrolysis that follow ligation of theTCR by peptide/MHC complexes. Native, mutant or derivatized SAgs areadministered to stimulate anergized T and/or NKT cells to becometumor-specific effector cells now fully reactive against tumors. Suchcells are used also in adoptive immunotherapy of cancer as described inExamples 7, 15, 16, 18-23. Nucleic acid constructs comprising DNAencoding SAg and SAg receptor are provided to reverse T cell anergy incancer patients.

Anergic T (or NKT) cells transfected with DNA encoding a SAg receptorexpress the receptor on the cell surface. Binding of exogenous SAg tothis receptor generates T cell activating signals, so that the activatedT cells can be used for adoptive immunotherapy.

DNA encoding a SAg peptide is transfected into cancer patients'anergized T and/or NKT cells. These DNA constructs also contain the ISS(described above). The T and/or NKT cell transfectants have revitalizedproliferative activity when stimulated by tumor-specific antigen andexogenous SAg. The cells are used for adoptive immunotherapy of cancer(Examples 7, 15, 16, 18-23).

Anergic T cells from cancer patients are transfected in vivo or invitro, resulting in a population of tumor reactive effector T cells invivo or ex vivo. The ex vivo transfected T cells are used for adoptiveimmune therapy as described in Example 15, 16, 18-23. In the case whereSAg receptor is expressed by transfected T cells, these cells areactivated by locally or systemically by SAgs to result in tumor-specificeffector cells.

Additional manipulations that assist in restoring responsiveness toanergic T cells include removal of the (T or NKT) cells from theimmunosuppressive microenvironment and transfer into tissue culture fora short period before stimulation with SAg. Furthermore, defectivesignaling in patient T or NKT cells may be reconstituted by transfectionwith DNA encoding CD3-2 or fyn that is either in a single constructwith, or cotransfected with, DNA encoding SAg and/or SAg receptor. Now,surface activation of the SAg receptor triggers CD3-signaling and T cellproliferation.

SAg activation of a T cell surface ganglioside (such as GD3) is alsoused to reverse T cell anergy in cancer patients. SAg coupled to GalCer,lipopolysaccharides or proteosomes are even more effective in activatingsuch anergized T cells. Coordinate activation of CD69 with phorbolesters in combination with SAg stimulation also reverses T cell anergy.

EXAMPLE 10 Tumor Specific Effector T or NKT Cells as Lymphoid Organoids

Tumor specific T and/or NKT effector cells (or hybridomas with suchcells) are prepared ex vivo in the form of a lymphoid organoid andimplanted into tumor-bearing hosts. The organoid consists of the tumorspecific lymphocytes either activated by SAgs, transfected to expressSAg alone or in combination with the other proteins or anti-tumormoieties described herein. The cells are encased in semi-permeablemembranes that allow for their progressive entry into the blood andlymphatics after implantation into the host. Such organoids areimplanted preferentially at sites adjacent to lymphatics or bloodvessels that drain organs or regions of known tumor involvement.However, they may also be implanted subcutaneously, intraperitoneally inaddition to intra-tumorally or adjacent to a tumor site. The advantageof the organoid is that it continuously provides proliferatingtumor-specific effector cells that recognize traffic to tumor sites in aphysiological manner. This approach avoids negative selection,functional deficiencies and storage problems associated with long termcultured cells.

Organoids are encased in macrocapsules, sheaths, rods, discs, orspherical dispersions or microcapsules. Microcapsules are made ofhydrogels such as polysaccharide alginate that are optionally coatedwith polyanions and again with alginate. Macrocapsule and vasculardevices consists of acrylonitrile-vinyl chloride copolymers or cellulosenitrate membranes. In one approach, scaffolds composed of syntheticpolymers serve as cell transplant devices. The polymers are degradableor non-degradable materials that disappear from the body after theyperform their function to obviate concerns about long-termbiocompatibility.

These devices serve as structural and functional tissue units by thetransplanted cells. The open system implants are designed so that thepolymer scaffold guides cell organization and growth and allowsdiffusion of nutrients and cells. The cell polymer matrix ispre-vascularized or becomes vascularized as the cell mass expands afterimplantation. Vascularization is induced naturally by the host orartificially by secretion of angiogenic factors from host cells.Optionally, the angiogenic proteins are genetically engineered into thehost T cells in vitro before implantation or in vivo before or afterimplantation.

To maintain or facilitate targeting of the cells to tumors or involvedorgans, the lymphocytes are transfected with DNA encoding polypeptidesthat enhance homing and trafficking ability to the sites of tumor burden(e.g., brain, liver, lung). The organoid lymphocytes express no CTLA sothat they may proliferate (in vitro and in vivo) without the need forexogenous IL2. Alternatively, cells are transformed to express herpessimplex virus thymidine kinase, making them susceptible to killing bygancyclovir. This curtails uncontrolled proliferation caused by theCTLA-4 deletion (or inhibition). Exogenous control of antitumor activityis achieved through the use of inducible promoters, such as thoseresponsive corticosteroids or metals.

EXAMPLE 11 Tumor Specific Effector Cells or Tumor Cells ExpressingProtein A, Protein A Domains and/or Angiostatin

It is desirable to express Fc receptors (FcR) or Ig VH3 domains on tumorcells to promote binding by immunoglobulins and enhance damage byantibody dependent cellular cytotoxicity. By introducing StaphylococcalProtein A, or its domains A-D into tumor cells which overexpress FcR andVH3 the tumor cells bind immunoglobulins (including those withαGalspecificity). Signaling of T cells occurs via high affinity binding toFcR (FcRI ) of protein A-IgG complexes; such binding bypasses the CD3-blockade in tumor bearing patients. These transfected tumor cells areuseful as a vaccine. Likewise, nucleic acids encoding protein A and itsdomains A-D are transfected into partially or fully anergized T or NKTcells of cancer patients. Exogenous immunoglobulins stimulate thegeneration of tumor-specific effector T or NKT cells which are used inadoptive immunotherapy (Examples 7, 15, 16, 18-23).

DNA encoding Staphylococcal protein A and its domain D areco-transfected into these tumor cells resulting in the joint surfaceexpression of: (1) protein A and FcR to which it binds and/or (2) domainD and Ig VH3 to which it binds. When DNA encoding protein A or domain D,fused to a signal sequences that route and anchor the protein A peptideto the tumor cell surface, is introduced into tumor cells, such tumorcells are excellent targets for parenterally administered SAgpolypeptides (particularly those for which no natural antibodies exist).Tumor cells expressing protein A and domain D and also expressing FcRson the cell surface, have heightened sensitivity to complement mediatedlysis. Tumor cells cotransfected to express protein A and Gal (byintroduction of the appropriate glycosyltransferase) are capable ofreacting with natural anti-Gal antibodies, Ig Fc fragments and Ig VH3domains, which stimulate an enhanced tumoricidal response.

Angiostatin is a circulating angiogenesis inhibitor which is 38-kDainternal fragment of (mouse) plasminogen that contains the first fourdisulfide-linked kringle domains. In vivo, angiostatin suppressesneovascularization in several traditional assays (chick chorioallantoicmembrane assay and mouse corneal assay). Proteases released by tumorcells cleave circulating plasminogen to generate angiostatin.Metalloelastase produced by tumor infiltrating macrophages generatedangiostatin production by murine Lewis lung carcinoma. In the presentinvention, nucleic acid encoding angiostatin (Cao Y et al., J. Clin.Invest. 101: 1055-1063, (1998)) are cotransfected into tumor cells withnucleic acid encoding SAg (as in Example 1). The tumor cellcotransfectants express and secrete SAg and angiostatin. Such cells areused directly as a preventative vaccine (Example 8) or as a therapeuticvaccine to treat established tumor including micrometastases. Methodsfor using these cells in vivo are in Examples 7, 12, 16, 18-23.

In addition, tumor cells are cotransfected to express angiostatin andprotein A (and/or its domains). Any nucleic acid construct shown inTable I may also be used in combination to transfect tumor cellstogether with protein A, its domains and angiostatin.

EXAMPLE 12 SAg Receptor

Colon carcinoma is used as the tissue source for the SEB receptor.Mixtures of different detergents at low concentrations are used. Theprotocol for screening detergents for solubilization of MAChRs isreadily adaptable to other receptor types. The membranes are suspendedat 5-10 pH 7.5, 20 mM Tris-HCl, pH 7.5, or 20 mM sodium phosphate, pH7.0-7.5. For screening purposes it is unnecessary to add complexproteolysis inhibitor cocktails. The presence of EDTA (1 mM) to inhibitcalcium-activated proteases and. of PMSF or benzamidine (0.1 mM) toinhibit serine proteases is sufficient. Mg2⁺⁺ (2 mM) is added. Themembranes are prelabelled with a radioligand in the presence and absenceof a suitable unlabelled ligand to determine the total and non-specificbinding. Non-specific binding is subtracted from total binding to obtainthe specific binding. A high enough concentration of labeled ligand tosaturate the binding site(10×K_(d)) is used, so that the bindingcapacity is measured. The unlabelled ligand is used at a concentrationof 1000×K_(d). The normal criteria for specific binding must befulfilled. The incubation is sufficient to reach equilibrium.Prelabelled membrane suspension (0.5 ml) is added to a series ofcentrifuge tubes a 4° C. An equal volume of detergent solution in thesame buffer is added to obtain a series of different final detergentconcentrations, e.g., 0, 0.1, 0.2, 0.5, 1.0, 2.0% w/v. The tubes aremixed and incubated for 60 min. at 4° C. Solubilization is assisted bystirring or mixing, e.g., with a rotating-wheel end-over-end mixer. Thetubes are centrifuged for 30-60 min at 100,000×g for 60 min. Forscreening, a lower speed spin, e.g., 10,000×g for 5 min (such as in amicrofuge) is acceptable. Supernatant, 0.2 ml, is applied to a 2 mlcolumn of Sephadex G50 equilibrated with the selected detergent at 0.1%.When the sample has run in, 2×0.2 ml of detergent-buffer is applied andthen the void volume fraction is eluted with 0.5 ml of detergent buffer.This procedure is carried out, the remaining material is removed, and 10ml of aqueous scintillation cocktail is added and the radioactivitycounted. Sephadex G50 is substituted for G50F for hydrophilic ligands,which do not partition into detergent micelles. This gives a more rapidseparation. The recovery of specifically bound ligand is calculated inabsolute terms:bound ligand=(dpm(total)−dpm(non-spec.)×5/(2220×spec. .act) pmol/ml

An aliquot of the pellets is resuspended and counted to calculaterecovery of unsolubilized receptors. The concentration of protein in thesolubilized supernatant is measured, for example, by measuring UVabsorbance at 280 nm against a detergent-buffer blank. (If necessary,the supernatant is diluted to get the absorbance on scale.) Proteinconcentration in the solution is approximately equal to the absorbanceat 280 nm. Alternatively, the Lowry method is used. The above steps arerepeated without first prelabeling the receptors in the membrane.Instead, the solubilized supernatant is incubated in the absence andpresence of labeled ligand. Again, concentration of the labeled ligandis used that saturates the binding sites. Incubation is carried out for2 h at 4° C., and the binding is assayed by gel filtration as above. Thepellet is resuspended and assayed for residual binding to check overallrecovery. The molecular size of the receptors in solubilizedpreparations is estimated by a combination of gel filtrationchromatography and sucrose density gradient centrifugation in H₂O andD₂O. Affinity chromatography is the principal method use forpurification of all of the receptors, combined with gel permeation HPLC,and ion exchange. SDS PAGE is carried out on the final product. Affinitychromatography is carried out using immobilized SEB, and the column iseluted with acid buffer or different concentrations and ionic strengthsof eluting buffer.

Determination of Amino-Acid and Oligonucleotide Sequences of SAgReceptors

Receptor material is eluted from the SDS-PAGE, and the N-terminal aminoacid sequence is determined. When free amino termini are not available,the purified receptor material must be subjected to partial hydrolysis.The specific cleavage of peptide bonds is performed with endoproteases,such as V8 protease or trypsin, or with chemicals such as cyanogenbromide(CNBR). The resulting peptides are separated by SDS-PAGE whenthey are over residues or by reverse phase HPLC. The peptides thusanalyzed are subjected to amino-acid sequence analysis with a gas phaseor solid phase sequencer.

Antibodies are raised against the peptides and the resultant antibodiesused to confirm that the peptide is a part of the receptor byimmunoprecipitation or Western blot.

To determine the full sequence of the receptor gene,oligodeoxynucleotide probes synthesized on the basis of peptidesequences are used to screen an appropriate cDNA library. Either amixture of relatively short oligonucleotides with all possible sequencesor a relatively short oligonucleotides with a sequence based on codonusage frequency is used. Genomic libraries as well as cDNA libraries arescreened to obtain genes for receptors and to deduce their amino acidsequence. The amino acid sequence deduced from the nucleotide sequenceis compared to the known sequences of other receptors. Among the usefulstructural information derived from the sequence analysis is thehydropathy profile. The presence of hydrophobic domains with a length ofapproximately 20 amino acids residues suggests that the regions aretransmembrane segments. Genomic or cDNA clones ligated into expressionvectors are used to transform suitable cell lines.

Alternatively, mRNA transcribed from these clones is injected intorecipient cells such as Xenopus oocytes. The expression of receptors inthese cells is confirmed by measuring ligand binding, reactivity of cellhomogenates or membrane preparations with antibodies or the responsesinduced by receptor agonists in recipient cells. The direct function ofthe receptors is elucidated by reconstituting purified receptors inphospholipid vesicles with or without other components. An additionalmethod is based on the isolation of cDNA or genomic clones for receptorswithout using purified receptors. The structure of receptors andcellular responses to them is examined using these clones. Substantialamounts of receptor material is produced from these clones. Monoclonalantibodies to the SAg receptors are used to screen clones for receptorsderived from cDNA libraries constructed with expression vectors.

Transfection of SAg receptor involves the ligation of the receptor geneinto an appropriate expression vector, transformation of a suitablebacterial host, and isolation of an individual bacterial colonycontaining the plasmid vector. The plasmid DNA is harvested from thelysed bacteria. The preferred method of purification of plasmid DNA foruse in transfections involves Triton-lysozyme equilibrium gradient. Thecells to be used in transfection are maintained in the log phase ofgrowth at all times. The calcium phosphate method is useful andefficient means for introduction of cloned genes in plasmid vectors intomammalian cells as described earlier in this document is preferred.However, the other methods given are useful as well. A partial list ofplasmid vectors and promoters suitable for transfection of culturedmammalian cell is given in Fraser, C. M., Expression of Receptor Genesin Cultured Cells in Receptor Biochemistry, A Practical Approach, Hulme,E. C., ed., Oxford University Press, pp. 263-275, 1993.

EXAMPLE 13 Avoiding Interference with SAg-Specific Antibodies

Naturally antibodies are found in mammals that are specific for the SAgmolecule (e.g., a Staph enterotoxin). Such antibodies bind and interferewith the SAg expressed and secreted by transfected cells. Suchantibodies also hinder therapeutic action of SAg infused directly (asnative protein, peptide or fusion protein).

It is desirable to neutralize or otherwise remove such before the cellsof this invention are administered to a subject. One way to achieve thisis to pre-treat the subject with antiidiotypic antibodies specific forthe variable region of SAg-specific antibodies. Another way is to infuseSAg peptides that represent the major immunogenic portions of theoverall protein. Alternatively, SAg is immobilized to a solid support bycovalent bonding and the blood or plasma is perfused extracorporeallythrough a device containing the immobilized protein, thereby removingthe antibodies by immunoadsorption. In another approach, SAg-expressingcells (prokaryotic or eukaryotic) preferably of host origin, or phagedisplays, are encapsulated and used as immunoadsorbents to bindscirculating SAg-specific antibodies. An organoid containing theseadsorbing cells is positioned subcutaneously or placed into thecirculation via catheter and then removed once the adsorption process iscomplete. Alginate encapsulated cells expressing SAg are preferred butother known modes of cell encapsulation may be used. Liposomes withsurface-bound SAg are another form of immunoabsorbent that are employedeither as an organoid or by direct injection.

Induction of Immunological Tolerance

The induction of tolerance to epitopes of the SAg molecule which inducea humoral antibody response would be desirable. The portion of the SEAmolecule which binds to natural antibodies is the linear sequence ofresidues 232-262. Immune tolerance is induced using this sequence by themethod of Dintzis et al., Proc. Natl. Acad. Sci. 89: 1113-1117 (1992).in which low molecular weight peptide arrays are administered topatients with circulating antibodies to enterotoxins. The peptides aredelivered parenterally or orally once weekly in doses of 1-500 mg/kg forthree to six weeks after which there is a reduction and disappearance ofcirculating antibody specific for the tolerogen.

After one or more of the foregoing treatments, native SAg or SAgconjugated to a monoclonal tumor-specific antibody and administered tothe host can now localize to tumor sites without diversion bycirculating SAg-specific antibodies.

Phage Displayed SAgs

Phage display technology may also be used neutralize circulatinganti-enterotoxin antibodies. The SAg and/or SAg receptor is expressed atthe surface of bacteriophage as a fusion protein with the gene VIIIprotein (gVIIIp). This phage-displayed SAg fusion protein retains theproperties of the natural protein. For this invention, the filamentousphage vector f88-4 which forms a fusion protein between the C terminusof the inserted gene product and the N terminus of gVIIIp is used. Thephage expressing SEA is injected intravenously into patients that havenatural antibodies to SEA. The amount of phage (transducing units)required to neutralize the circulating pool of antibodies ispredetermined by antigen binding inhibition assay. The number oftransducing units required to neutralize the pool of circulating SEAspecific antibodies is administered intravenously. Shortly after thisinjection, the host is ready for treatment with active SEA which is nolonger hindered from finding its “target,” ie., enterotoxin receptorsexpressed by tumor cells or T cells.

SEA clone pKH-X35 is employed. PCR with Vent Polymerase 9NEB is used tomutate the 5′- and 3′-ends of the SEA gene for cloning into f88-4. Theconstruct is as follows. The 5′ oligonucleotide used is5′-CTCCAAGCTTTGVCCAGCGAGAAAAGCGAAG-3′. Two 3′ oligonucleotide primersare used. For the construct with the five amino acid linker between SEAand gVIIIp (SEA L), the primer5′-GCCTCCTGCAGATCCACCGCCTCCGGATGT-ATATAAATATATATC-3′ and for thenon-linker version (SEA-P); 5′-GCCTCCTGCAGATGTATATAAATATATATC-3′ areused. The two SEA PCR products are cut with HindIII and PstI and clonedinto f88-4. They are transformed by electroporation into E. coli strainDH5a and sequenced. Phage are produced by growing the transformedbacteria overnight in 0.5 L of broth with 20 mg/ml tetracycline. Theculture is pelleted twice (800×g for 15 min) and the phage precipitatedout of the cleared supernatant by the addition of 0.15 vols. of PEG/NaClsolution (17% PEG 8000, 19% NaCl in water). After incubation at 4° C.for 2 hours, the phage are resuspended in TBS and sterile-filteredthrough a 0.22-m membrane. Phage are selected by the micropanningtechnique and by cell binding. Binding to antibody is assessed byattaching mAb to the surface of 96-well ELISA plates, blocking with 1%BSA, incubating with 100 mg/ml of SEA or PBS as a control and thenincubating with the various phage preparations for >2 hours at 4° C. Thephage is then eluted with 0.1 M HCl pH 2 (adjusted with glycine) for 10minutes, neutralized and used to infect starved E. coli MC 10161 F′ Kan.The infected bacteria are then spread on tetracycline (20 mg/ml) LB agarplates. After overnight culture tetracycline resistant colonies arecounted representing the number of transducing units (TU) recovered. Todetermine the number of SEA-bearing phage among thetetracycline-resistant colonies, colony blotting is performed bystandard techniques probing with a ³²P-labeled SEA probe. An antibodybased variant of this technique is involves probing with a rabbitanti-SEA serum as for Western blots.

Chimeric Enterotoxins

Likewise, hybrid or chimeric SAgs that are non-immunogenic are used tostimulate cells. When these molecules are injected into hosts that havenatural antibodies, they are not rapidly eliminated from thecirculation. Such chimeric molecules lacking the binding site fornatural antibodies preserve the T cell mitogenic and cytokine-inducingproperties of the native SAg. A peptide sequence from another SAg towhich antibodies do not exist is substituted using genetic orbiochemical methods well known in the art. This is particularly usefulin the case of enterotoxins such as SEB or SEA to which a largepercentage of humans have naturally occurring circulating antibodies.The antibody binding region of these molecules near the C terminalregions is delineated. The substitution of the antibody bindingsequences in SEA or SEB for sequences from SEE or SED to which a verysmall number of humans have circulating antibodies markedly enhances thetumor killing efficacy of the injected chimeric enterotoxins.

A hybrid molecule consisting of a 26 amino acid peptide corresponding tothe N-terminal portion of SEA, the loop structure of SEA, a conservedmid-molecular sequence of SEA and SEB, and a C terminal sequence of SEBwas synthesized in collaboration with Multi-Peptide Systems, La Jolla,Calif. Peptides were prepared using a variation of Merrifield's originalsolid phase procedure in conjunction with simultaneous multiple peptidesynthesis using t-Boc chemistries. Peptides were cleaved from the resinsusing simultaneous liquid HF cleavage. The cleared peptides were thenextracted with acetic acid and ethyl ether and lyophilized. Reversephase HPLC analysis and mass spectral analysis revealed a single majorpeak with the molecular weight corresponding closely to theoretical.

Synthetic SAgs

Amino acid sequences of SEA and SEB known to be involved in theinteraction with the TCR and MHC class II molecules are retained. Theloop structure of SEA is retained because it is devoid of histidinemoieties that are associated with the emetic response. Residues 1-10 ofthe N-terminal region of SEA are retained because they have MHC class IIbinding activity. The loop structure of SEA is retained because it andassociated disulfide linkages are considered to be important for Tlymphocyte mitogenicity, stabilization of the molecule, and resistanceto in vivo degradation. A conserved sequence in the central portion ofSEA and SEB adjacent to the disulfide loop (amino acids 107-114) wasretained. Histidine moieties are deleted from the molecule because oftheir association with the emetic response.

Synthesis Procedure

The preparation of all peptides was carried out using a variation ofMerrifield's original solid phase procedures in conjunction with themethod of Simultaneous Multiple Peptide Synthesis using t-Bocchemistries (Merrifield R B I, J. Amer. Chem. Soc. 85:2149-2154 (1963));Houghten R A, Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985); andHoughten R A et al., Intact. J. Peptide Protein Res. 27:673-678 (1985)).

4-methylbenzhydrylamine (mBHA) and phenylacetamidomethyl (PAM) resinswere purchased from Advanced Chemtech (Louisville, Ky.) and Bachem(Torrance, Calif.), respectively. All of the amino acids contained thet-butyloxycarbonyl (t-Boc)-amino protecting group and were purchasedfrom Bachem. The side chain protecting groups included benzyl(threonine, serine and glutamic acid), chlorobenzyloxycarbonyl (lysine),bromobenzyloxycarbonyl (tyrosine), cyclohexyl (aspartic acid), p-toluenesulfonyl (arginine), formyl (tryptophan), methyl benzyl (cysteine), anddinitrophenyl or benzyloxycarbonyl (histidine). Cysteine with the HFstable acetamidomethyl (ACM) protecting group was used, upon request,for internal cysteines. Each lot of amino acid derivative was tested bymelting point analysis. Reagent grade methylene chloride (CH₂Cl₂),isopropanol (IPA), and dimethylformamide (DMF) were obtained from FisherScientific (Tustin, Calif.). diisopropylcarbodiimide (DIPCDI) anddiisopropylethylamine (DIEA) were purchased from Chem Impex (Wood Dale,Ill.). Trifluoroacetic acid was purchased from Halocarbon (Hackensack,N.J.).

The appropriate resin, mBHA for C-terminal amides and PAM for C-terminalacids, was weighed with a Mettler AE 240 balance (Highstown, N.J.) intoseparate polypropylene mesh (74 mm) packets which had been pre-sealed on3 of 4 sides using a TSW TISH-300 Impulse Sealer (San Diego Bag andSupply; San Diego, Calif.). Each packet was also pre-labeled with areference code using a KOH I NOOR Rapidograph pen with graphite basedink to allow them to be easily identified during resin addition andduring the synthesis process. Each packet was then careftully sealedcompletely to make sure there would be no resin leakage. All the resincontaining packets (up to 150) were then placed in a common Nalgenebottle. Enough CH₂Cl₂ to cover all the packets was then added to thebottle, which was then capped and vigorously shaken for 30 seconds on anEberbach Shaker (Fisher Scientific; Tustin, Calif.) to wash and swellthe resin. The CH₂Cl₂ solution was then removed. All subsequent stepsinvolved the addition of enough solvent to cover all the packets andvigorous shaking to ensure adequate solvent transfer. The N--t-Boc wasremoved by acidolysis using a solution of 55% TFA in CH₂Cl₂ for 30minutes, leaving the TFA salt of the α-amino group. The TFA washsolution was then removed. The packets were then washed for 1 min withCH₂Cl₂ (2×), IPA (2×) and CH₂Cl₂ (2×) to squeeze out excess TFA and toprepare for neutralization. The TFA salt was neutralized by washing thepackets three times with 5% DIEA in CH₂Cl₂ for two minutes each. Thiswas followed by two washes with CH₂Cl₂ to remove excess base.

The resin packets were then removed from the common Nalgene bottle andsorted according to computer generated checklists in preparation forcoupling. This was double checked to ensure the packets were added tothe correct amino acid solution. The packets were then added to bottlescontaining the appropriate 0.2 M amino acid in CH₂Cl₂ and/or DMFdepending on solubility. These solutions were also prepared usingcomputer generated information. An equal volume of 0.2 M DIPCDI was thenadded to activate the coupling reaction. The bottles were then shakenfor one hour to ensure complete coupling. At completion, the reactionsolution was discarded and the packets were washed with DMF for 1 min toremove excess amino acid and the by-product, diisopropylurea. A finalCH₂Cl₂ wash as then used to remove DMF. The packets were then removedfrom their individual coupling bottles and placed back into the commonNalgene bottle. The peptides were then completed by repeating the sameprocedure while substituting for the appropriate amino acid at thecoupling juncture. The packets were then taken through a finalacidolysis along with subsequent CH₂Cl₂, IPA and CH₂Cl₂ washes to leavethe peptides in the TFA salt form. The packets were then dried inpreparation for the next process.

Final side chain deprotection and cleavage of the anchored peptide fromthe resin was achieved through simultaneous liquid HF cleavage (HoughtenR A et al., supra.

Gaseous N2, HF, and argon were acquired from Air Products (San Diego,Calif.). Anisole was purchased from Aldrich Chemical Co. (Milwaukee,Wis.). Acetic acid (HOAc) and ethyl ether were purchased from FisherScientific (Tustin, Calif.). Each packet along with a Teflon coated stirbar was placed into an individual reaction vessel of a multi-vesselhydrogen fluoride apparatus (Multiple Peptide Systems; San Diego,Calif.). An amount of anisole equaling 7.5% of the expected volume of HFwas then added to act as a carbonium ion scavenger. The reaction tubeswere lubricated with vacuum grease at the point where each contacts theapparatus and sealed onto the HF system. The system was then purged withN₂ while cooling the reaction vessels to −70° C. using an acetone/dryice bath. HF (g) was condensed to the desired level and temperatureelevated to −10° C. using ice and water. The reaction was allowed toproceed for 90 minutes with the temperature slowly rising from −10° C.to 0° C. HF was removed using a strong flow of N₂ for 90 minutesfollowed by the use of aspirator vacuum for 60 minutes while maintainingthe temperature at 0° C. The reaction vessels were then removed from theapparatus and capped. The residual anisole was removed with two ethylether washes. The peptide was then extracted with two 10% HOAc washes. A50 ml sample of the crude peptide was taken and run on an analyticalBeckman 338 Gradient HPLC System (Palo Alto, Calif.) using a Vydac C18column to profile the initial purity of the compound. The crude peptidewas then lyophilized twice on a Virtis Freezemobile 24 Lyophilizer,weighed and stored under argon.

Analytical RP-HPLC was used to determine the homogeneity and approximateelution conditions of the peptides produced. HPLC grade acetonitrile(ACN) was purchased from Fisher Scientific (Tustin, Calif.). HPLC gradeTFA was obtained from Pierce Chemicals (Rockford, Ill.). RP-HPLCanalysis was carried out on a Beckman 338 Gradient HPLC system (PaloAlto, Calif.) equipped with a BioRad AS-100 autosampler and a ShimadzuCR4A integrator. The column used for all analyses this quarter was aVydac C-18 column (4.6×250 mm). The solvent system used was 0.05%aqueous TFA (A) and 0.05% TFA in ACN (B) with a flow rate of 1 ml/min.Absorbance was measured at 215 nm. Most peptides were analyzed using thefollowing special gradient; 5.60% (B) in 28 minutes. Hydrophobicpeptides were analyzed using the following special gradient: 5-40% (B)in 9 minutes, 40-90% (B) for 10 additional minutes, 95% (B) for the last9 minutes.

Analytical data was reviewed. The product peak was identified and markedbased upon knowledge of common impurities and the use of predicted HPLCretention times.

Peptides that did not meet normal purity requirements for crude materialwere purified using preparative RP-HPLC techniques. HPLC gradeacetonitrile (CAN) was purchased from Fisher Scientific (Tustin,Calif.). HPLC grade TFA was obtained from Pierce Chemicals (Rockford,Ill.). Purification was carried out on a Waters Delta Prep 3000 with aPreparative Waters Prep Pak Module Radial Compression C18 column (5cm×25 cm, 10-20 m). The solvent system used was 0.05% aqueous TFA (A)and 0.05% TFA in ACN (B). The crude peptides were solubilized in anHOAc/H₂O mixture and injected onto the column with 0.25% to 0.50% ACNper minute linear gradient. The absorbance was measured at 230 nm and 40ml fractions were collected upon elution with an ISO Fraction Collector(Lincoln, Nebr.). The preparative profile was reviewed and selectedfractions were analyzed by analytical RP-HPLC. The analytical data wasreviewed and fractions were combined and lyophilized. The lyophilizedmaterial was weighed, sampled for a final analytical RP-HPLC analysisand stored under argon in powder form. This process was repeated if thepurity level attained was not sufficient. Mass spectral analysis wasused to determine the molecular weight of the peptides produced. 95%ethanol was purchased from Fisher Scientific (Tustin, Calif.). HPLCgrade TFA was obtained from Pierce Chemicals (Rockford, Ill.).Nitrocellulose matrices (targets) were purchased from Applied Biosystems(Foster City, Calif.).

The samples were solubilized in a 1:1 solution of 95% ethanol and 0.1%TFA (aqueous). The samples were applied to a nitrocellulose matrix(Target). The mass spectra were obtained using an ABI Bio-Ion 20 MassSpectrometer (Foster City, Calif.). The apparatus makes use of plasmadesorption ionization via a Cf252 source. The ionized molecules are thenanalyzed via time-of flight. An accelerating voltage of 15,000 V is usedto accelerate the particles.

The Protocol for Intramolecular Disulfide Bridge:

Dissolve crude peptide (300-500 mg) in 200 ml of deoxygenated water andadjust the pH to 8.5 using NH₄OH 28%=Solution A. Note: If the peptide isnot very soluble in water, some MeOH can be added.

Dissolve 0.5 g K₃Fe(CN)6 in 200 ml of deoxygenated water and adjust thepH to 8.5 using NH4OH 28%=Solution B. Note: 0.5 g K₃Fe(CN)₆ is anaverage value for 500 mg of a 10 mer peptide. The excess of K₃Fe(CN)₆should be approximately 3×. It can be adjusted.

Solution A is then dropped slowly into solution B over a 2 hour period.The mixture is then allowed to react, for an additional 1 hour withstirring. The pH is then adjusted to 4.0-4.5 with 10% ACTH. Thissolution is injected directly into a preparative RP-HPLC. The major peakis then collected. This “pseudo dilution” technique favors theintramolecular disulfide. Therefore, the major peak is the cyclicproduct.

The chimeric enterotoxin molecule was tested in normal rabbits andrabbits with established VX2 carcinoma. It was administeredintravenously and peripherally with adjuvant. The chimeric molecule (1mg/ml) was diluted initially in 1 ml of sterile H₂O. When the solutionwas clear, 9 ml of normal saline was added. The solution was filteredthrough a 0.45 m filter and stored in 0.5-1 ml aliquots. Dosage rangedfrom 2.6-5.0 mg/kg and was described over 3 minutes via the lateral earvein in a volume of 0.05 ml diluted further in 1.0 ml of 0.15 M NaCl:

The i.v. line was then washed with 3 ml of 0.15M NaCl. In two animals,the temperature rose only 0.3 F over the ensuing 24 hours and there wasno discernible toxicity over the ensuing 14 days of observation. Oneanimal was described a second dose of the chimeric molecule in pluronicacid triblock adjuvant. This was described in a dose of 8.5 mgsubcutaneously in each thigh with a total dose 5 mg/kg. The pluronicacid triblock preparation was prepared as follows: 4.23 cc PBS; 0.017 ccTween; 0.05 cc Squalene; and 0.25 cc Pluronic. The PBS and Tween weremixed first then squalene was added followed by pluronic acid. The totalmixture was vortexed for 3-4 minutes. Two ml of above plus 0.34 ml ofthe chimeric protein (34 mg) plus 1.66 cc PBS were added to the mixture.The mixture was vortexed vigorously for 1-2 minutes. One ml was injectedinto each thigh (total vol. injected was 0.17 ml or 17 mg protein or 5mg/kg).

For nearly 5 weeks after injection, no adverse effects were noted. Thetumor showed slow, but progressive growth over this period of time. Todate, the chimeric enterotoxin molecule appears to be safe in animalsand no untoward side effects were demonstrated. The adjuvant used forthese studies was the pluronic acid triblock copolymer which has beenused to boost the immune response to various antigens in animal modelsand which is under testing at this point in humans with hepatitis andherpes simplex infections. Other adjuvants including those prepared inwater and oil emulsion and aluminum hydroxide to administer various SAgsin vivo to tumor bearing rabbits were also used.

Additionally, enterotoxins such as SEE, SED, SEC, and TSST-1 are used toprepare hybrid molecules containing amino acid sequences and homologousto the enterotoxin family of molecules. To this extent, mammary tumorvirus sequences, heat shock proteins, stress peptides, Mycoplasma andmycobacterial antigens, and minor lymphocyte stimulating loci bearingtumoricidal structural homology to the enterotoxin family are useful asanti-tumor agents. Hybrid enterotoxins and other sequences homologous tothe native enterotoxins are immobilized or polymerized genetically orbiochemically to produce the repeating units and stoichiometry requiredfor (a) binding of accessory cells to T lymphocytes and (b) activationof T lymphocytes.

EXAMPLE 14 Pharmaceutical Compositions and their Manufacture

The pharmaceutical compositions may be in the form of a lyophilizedparticulate material, a sterile or aseptically produced solution, atablet, an ampoule, etc. Vehicles such as water (preferably buffered toa physiological pH such as PBS or other inert solid or liquid materialmay be present. In general, the compositions are prepared by being mixedwith or dissolved in, bound to or otherwise combined with one of morewater-insoluble or water-soluble aqueous or non aqueous vehicles, ifnecessary together with suitable additives and adjuvants. It isimperative that the vehicles and conditions shall not adversely affectthe activity of the conjugate. Water as such is comprised within theexpression vehicles. A suitable therapeutic composition is used in thetreatment of cancer of any kind including but not limited to carcinomas,sarcomas, lymphomas, leukemias and comprises a combination of:

-   (1) a recombinant DNA molecule encoding SAg in combination with,    preferably fused with, another recombinant DNA sequence encoding    another protein;-   (2) a recombinant DNA molecule encoding SAg-in combination with    another peptide or polypeptide; or-   (3) a recombinant DNA molecule encoding a protein other than a SAg    in combination with a SAg peptide or polypeptide.

These compositions that may comprise more than one components areadministered together or sequentially and they may be combined(separately or together) with a delivery vehicle, preferably liposomesas disclosed herein. Upon entering its intended or targeted cells, thetherapeutic composition leads to the production of SAg and a secondprotein that may result in (a) apoptosis of the cancer cell and (b) withor without such apoptosis, the activation of effector cells of theimmune system, including any or all of the following: cytotoxic T cells,NKT cells, NK cells, T helper cells and macrophages. The presenttherapeutic compositions are useful for the treatment of cancers, bothprimary tumors and tumor metastases.

Use of the present therapeutic composition overcomes the disadvantagesof traditional treatments for metastatic cancer. For example.compositions of the present invention can target dispersed metastaticcancer cells that cannot be treated using surgery. In addition,administration of such compositions is not accompanied by the harmfulside effects of conventional chemotherapy and radiotherapy.

A therapeutic composition also comprises a pharmaceutically acceptablecarrier defined as any substance suitable as a vehicle for delivering anucleic acid molecule (alone or in some combination with a protein) to asuitable in vivo or in vitro site. Preferred carriers are capable ofmaintaining DNA in a form that is capable of entering the target celland being expressed by the cell. Preferred carriers include: (1) thosethat transport, but do not specifically target a nucleic acid moleculeto a cell (referred to herein as “non-targeting carriers”); and (2)those that deliver a nucleic acid molecule to a specific site in ananimal or a specific cell (“targeting carriers”). Examples ofnon-targeting carriers are water, phosphate buffered saline (PBS),Ringer's solution, dextrose solution, serum-containing solutions, Hank'sbalanced salt solution, other aqueous, physiologically balancedsolutions, oils, esters and glycols. Aqueous carriers can containsuitable additional substances which enhance chemical stability andisotonicity, such as sodium acetate, sodium chloride, sodium lactate,potassium chloride, calcium chloride, and other substances used toproduce phosphate buffer, Tris buffer, and bicarbonate buffer andpreservatives, such as thimerosal, m- and o-cresol, formalin and benzylalcohol.

Preferred substances for aerosol delivery include surfactant substancessuch as esters or partial esters of fatty acids containing from about6-22 carbon atoms. Examples are esters of caproic, octanoic. lauric,palmitic, stearic, linoleic, linolenic, olesteric, and oleic acids.

Other carriers can include metal particles (e.g., colloidal goldparticles) for use with, for example, a biolistic gun through the skin.

Therapeutic compositions of the present invention can be sterilized byconventional methods and may be lyophilized.

The compositions of the present invention are delivered using a deliveryvehicle that can be modified to target a particular site in a subject.Suitable targeting agents include ligands capable of selectively (i.e.,specifically) binding to another molecule at a particular site. Examplesare antibodies, antigens, receptors and receptor ligands. For example,an antibody specific for an antigen on the surface of a cancer cell canbe placed on the outer surface of a liposome delivery vehicle to targetthe liposome to the cancer cell. By manipulating the chemicalformulation of the lipid portion of a liposome preparation, it ispossible to modulate its extracellular or intracellular targeting. Forexample, the charge of the lipid bilayer of a liposome surface can bevaried chemically to promote fusion with cells having particular chargecharacteristics. Preferred liposomes comprise a compound that targetsthe liposome to a tumor cell, such as a ligand on the outer surface ofthe liposome that binds a molecule on the tumor cell surface.

Although the DNA constructs of the present invention can be administeredin naked form, a liposome is a preferred vehicle for delivery in vivo. Aliposome can remain stable in an animal for a sufficient amount of time,at least about 30 minutes, more preferably for at least about 1 hour andeven more preferably for at least about 24 hours, to deliver a nucleicacid molecule to a desired site. A liposome of the present inventioncomprises a lipid composition that can fuse with the plasma membrane ofthe targeted cell to deliver the encapsulated nucleic acid molecule intoa cell. Preferably, the liposomes' transfection efficiency is about 0.5μg DNA per 16 nmol of liposome delivered to about 10⁶ cells, morepreferably about 1.0 μg DNA per 16 nmol of liposome delivered to about10⁶ cells, and even more preferably about 2.0 μg DNA per 16 nmol ofliposome delivered to about 10⁶ cells.

For use in the present invention, any liposome that is used inart-recognized gene delivery methods is appropriate. Preferred liposomeshave a polycationic lipid composition and/or a cholesterol backboneconjugated to polyethylene glycol. Complexing a liposome with nucleicacids for uses described herein is achieved using conventional methods.A suitable concentration of DNA to be added to a liposome preparation aconcentration that is effective for delivering a sufficient amount ofDNA molecules to a cell so that the cell can produce sufficient SAgand/or a other transduced protein to induce tumoricidal activity or tostimulate or regulate effector cells in a desired manner. Preferably,between about 0.1 μg and 10 μg of DNA is combined with about 8 nmolliposomes; more preferably, between about 0.5 μg and 5 μg of DNA is usedeven more preferably, about 1.0 μg of DNA is combined with about 8 nmolliposomes.

Another preferred delivery system is the sickled erythrocyte containingthe nucleic acids of choice a given in Example 6. The sicklederythorcytes undergo ABO and RH phenotyping to select compatible cellsfor delivery. The cells are delivered intravenously or intrarterially ina blood vessel perfusing a specific tumor site or organ e.g. carotidartery, portal vein, femoral artery etc. over the same amount of timerequired for the infusion of a conventional blood transfusion. Thequantity of cells to be administered in any one treatment would rangefrom one tenth to one half of a full unit of blood. The treatments aregenerally given every three days for a total of twelve treaments.However, the treatment schedule is flexible and may be given for alonger of shorter duration depending upon the patients response.

Another preferred delivery vehicle is a recombinant virus particle, forexample, in the form of a vaccine. A recombinant virus vaccine of thepresent invention includes the DNA encoding the therapeutic compositionpackaged in a viral coat that allows entrance of the transducing DNAinto a cell and its expression. A number of recombinant virus particlescan be used, for example, alphaviruses, poxviruses, adenoviruses,herpesviruses, arena virus and retroviruses. Also useful as a deliveryvehicle is a “recombinant cell vaccine,” preferably tumor vaccines, inwhich allogeneic (though histocompatible) or autologous tumor cells aretransfected with a DNA preparation encoding the therapeutic proteins orpeptides to be expressed. The cells are preferably irradiated and thenadministered to a patient by any of a number of known injection routes.

The therapeutic compositions that are administered by “tumor cellvaccine,” includes the recombinant molecules without carrier. Treatmentwith tumor cell vaccines is useful for primary or localized tumors aswell as metastases. When used to treat metastatic cancer, which includesprevention of further metastatic disease, as well as, the cure existingmetastatic disease.

As used herein, the term “treating” a disease includes alleviating thedisease or any of its symptoms and/or preventing the development of asecondary disease resulting from the occurrence of the initial disease.

An “effective treatment protocol” includes a suitable and effective doseof an agent being administered to a subject, given by a suitable routeand mode of administration to achieve its intended effect in treating adisease.

Effective doses and modes of administration for a given disease can bedetermined by conventional methods and include, for example, determiningsurvival rates, side effects (i.e., toxicity) and qualitative orquantitative, objective or subjective, evaluation of disease progressionor regression. In particular, the effectiveness of a dose regimen andmode of administration of a therapeutic composition of the presentinvention to treat cancer can be determined by assessing response rates.A “response rate” is defmed as the percentage of treated subjects thatresponds with either partial or complete remission. Remission can bedetermined by, for example, measuring tumor size or by microscopicexamination of a tissue sample for the presence of cancer cells.

In the treatment of cancer, a suitable single dose can vary dependingupon the specific type of cancer and whether the cancer is a primarytumor or a metastatic form. One of skill in the art can test doses of atherapeutic composition suitable for direct injection to determineappropriate single doses for systemic administration, taking intoaccount the usual subject parameters such as size and weight. Aneffective anti-tumor single dose of a therapeutic recombinant DNAmolecule or combination thereof is an amount sufficient amount to resultin reduction, and preferably elimination, of the tumor after the DNAmolecule or combination has transfected cells at or near the tumor site.

A preferred single dose of SAg-encoding DNA molecule or fusion productthereof is an amount that, when transfected into a target cellpopulation, leads to the production of SAg in an amount, per transfectedcell, ranging from about 250 femtograms (fg) to about 1 μg, preferablyfrom about 500 fg to about 500 pg and more preferably from about 1 pg toabout 100 pg.

When the SAg-encoding DNA is combined with a second DNA moleculeencoding a second protein product, an effective single dose of a thesecond DNA molecule is an amount that when transfected into a targetcell population leads to the production of the second protein product inan amount, per transfected cell, ranging from about 10 fg to about 1 ng,more preferably from about 100 fg to about 750 pg.

An effective cancer-treating single dose of SAg-encoding DNA and asecond DNA molecule encoding a second protein when administered to asubject using a non-targeting carrier, is an amount capable of reducing,and preferably eliminating, the primary or metastatic tumor followingtransfection by the recombinant molecules of cells at or near the tumorsite. A preferred single dose of such a therapeutic composition is fromabout 100 μg to about 4 mg of total recombinant DNA, more preferablyfrom about 200 μg to about 2 mg, most preferably from about 200 μg toabout 800 μg of total recombinant molecules. A preferred single dose ofliposome-complexed, SAg-encoding DNA, is from about 100 μg of total DNAper 800 nmol of liposome to about 4 mg of total DNA molecules per 32μmol of liposome, more preferably from about 200 μg per 1.6 μmol ofliposome to about 3 mg of total recombinant DNA per 24 μmol of liposome,and even more preferably from about 400 μg per 3.2 μmol of liposome toabout 2 mg per 16 μmol of liposome.

One of skill in the art recognizes that the number of doses requireddepends upon the extent of disease and the response of an individual totreatment. Thus, according to this invention, an effective number ofdoses includes any number required to cause regression of primary ormetastatic disease.

A preferred treatment protocol comprises monthly administrations ofsingle doses (as described above) for up to about 1 year. An effectivenumber of doses (per individual) of a SAg-encoding DNA molecule and asecond DNA molecule encoding a second protein, when administered in anon-targeting carrier or when complexed with liposomes, is from about 1to about 10 dosings, preferably from about 2 to about 8 dosings, andeven more preferably from about 3 to about 5 dosings. Preferably, suchdosings are administered about once every 2 weeks until signs ofremission appear, followed by about once a month until the disease isgone.

The therapeutic compositions can be administered by any of a variety ofmodes and routes, including but not limited to, local administrationinto a site in the subject animal, which site contains abnormal cells tobe destroyed. An example is the local injection within the area of atumor or a lesion. Another example is systemic administration.

Therapeutic compositions that are best delivered by local administrationinclude recombinant DNA molecules

-   -   (a) in a non-targeting carrier (e.g., “naked” DNA molecules as        taught in Wolff K et al., 1990, Science 247, 1465-1468); and    -   (b) complexed to a delivery vehicle.

Suitable delivery vehicles for local administration include liposomes,and may further comprise ligands that target the vehicle to a particularsite.

A preferred mode of local administration is by direct injection. Directinjection techniques are particularly useful for injecting thecomposition into a cellular or tissue mass such as a tumor mass or agranuloma mass that has been induced by a pathogen. Thus, the presentrecombinant DNA molecule complexed with a delivery vehicle is preferablyinjected directly into, or locally in the area of, a tumor mass or asingle cancer cell.

The present composition may also be administered in or around a surgicalwound. For example, a patient undergoes surgery to remove a tumor. Uponremoval of the tumor, the therapeutic composition is coated on thesurface of tissue inside the wound or injected into areas of tissueinside the wound. Such local administration will treat cancer cells thatwere not successfully removed by the surgical procedure, as well asprevent recurrence of the primary tumor or development of a secondarytumor in the surgical area.

Therapeutic compositions that are best delivered by systemicadministration include recombinant DNA molecules complexed to a tumorbinding ligand or a ligand that binds to the tumor vasculature orstroma. Examples are antibodies, antigens, receptor, receptor ligand ora targeted delivery vehicle as disclosed herein. These delivery vehiclesmay be liposomes into which are incorporated targeting ligands,preferably ligands that targeting the vehicle to the site of tumor cellsor another type of lesion. For cancer treatment, ligands thatselectively bind to cancer cells, or to cells within the area of acancer cell, are preferred. Systemic administration is used to treatprimary or localized tumors and, in particular, tumor metastases whereinthe cancer cells are dispersed. Systemic administration is advantageouswhen targeting cancer in organs, especially those difficult to reach fordirect injection, (e.g., heart, spleen, lung or liver).

Preferred modes and routes of systemic administration includeintravenous injection and aerosol, oral and percutaneous (topical)delivery. Intravenous injection methods and aerosol delivery areperformed conventionally. Oral delivery is achieved preferably bycomplexing the therapeutic composition to a carrier capable ofwithstanding degradation by digestive enzymes in the subject's digestivesystem. Examples of such carriers, includes plastic capsules or tabletsas are known in the art. For topical delivery, the therapeuticcomposition is mixed with a lipophilic reagent (e.g., DMSO) that canpass into the skin.

The therapeutic compositions and methods of the present invention areintended for animals, preferably mammals and birds, in particular housepets, farm animals and zoo animals as these terms are generallyunderstood. By “farm animals” are intended animals that are eaten orthose that produce useful products (e.g., wool-producing sheep).Examples of preferred animal subjects to be treated are dogs, cats,sheep, cattle, horses and pigs. The present compositions and methods areeffective in inbred and outbred animal species. Most preferably, theanimal is a human.

Another component useful in combination with the therapeutic nucleicacids of this invention is an adjuvant suited for use with a nucleicacid-based vaccine. Examples of adjuvant-containing compositions include

-   1) SAg-encoding DNA and a second DNA encoding a recombinant protein;    or-   2) SAg-encoding DNA combined with another peptide or polypeptide; or-   3) DNA encoding a second recombinant protein and a SAg peptide or    polypeptide.

As indicated above, effective doses of a SAg-encoding DNA combined witha second DNA molecule, or a vaccine nucleic acid molecule are determinedconventionally by those skilled in the art. One measure of an effectivedose is that produces a sufficient amount of SAg and second protein tostimulate effector cell immunity in a manner that enhances theeffectiveness of the vaccine. Adjuvants of the present invention areparticularly suited for use in humans because many traditional adjuvants(e.g., Freund's adjuvant and other bacterial cell wall components) aretoxic whereas others are relatively ineffective (e.g., aluminum-basedsalts and calcium-based salts).

EXAMPLE 15 General Procedures for In Vivo and Ex Vivo Sensitization toProduce Tumor Specific Effector Cells for Adoptive Immunotherapy

Tumor growth is initiated by subcutaneous inoculation of mice on bothflanks with 1.5×10⁶ tumor cells suspended in 0.05 ml of HBSS. After 9-12days of tumor growth (approximately 8 mm in diameter), tumor-draininginguinal LN are removed sterilely. Lymphocyte suspensions are preparedby teasing LN with needles followed by pressing with the blunt end of a10-ml plastic syringe in HBSS. Tumor draining LN cells are stimulated invitro in a two-step procedure. Briefly, 4×10⁶ LN cells in 2 ml ofcomplete medium (CM) containing the SAg constructs are incubated in awell of 24-well plates at 37° C. in a 5% CO₂ atmosphere for 2 days. CMconsisted of RPMI 1640 medium supplemented with 10% heat-inactivatedFCS, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 2 mM freshlyprepared L-glutamine. 100 μg/ml streptomycin, 100U/ml penicillin, a 50mg/ml gentamycin, 0.55 mg/ml fungizone (all from GIBCO, Grand Island,N.Y.) and 5×10⁻⁵M 2-mercaptoethanol (Sigma). The cells were harvested,then washed and further cultured a 3×10⁵/well in 2 ml of CM with IL-2.After 3-day incubation in IL-2, the cells are collected and counted todetermine the degree of proliferation. Finally, the cells are suspendedin appropriate media for flow cytometric analysis, evaluation ofcytotoxicity and lymphokine secretion, or for adoptive immunotherapy.

EXAMPLE 16 General Adoptive Immunotherapy Protocol

Mice are injected with 2 to 3×10⁵ syngeneic tumor cells suspended in 1ml of HBSS to initiate pulmonary metastases. On day 3, activated cellsare given i.v. at numbers indicated generally 10⁶-10⁷. In someinstances, mice are also treated with 15,000 U IL2 in 0.5 ml HBSS twicedaily for 4 consecutive days to promote the in vivo function andsurvival of the activated cells. On day 20 or 21, all mice arerandomized, killed and metastatic tumor nodules on the surface of thelungs enumerated as previously described. If pulmonary metastasesexceeded 250, this number is arbitrarily assigned for statisticalanalysis. The significance of differences in metastases numbers betweenexperimental group is determined by the Wilcoxon rank sum test. Twosided p values of <0.1 are considered significant. Each experimentalgroup consists of at least five mice and no animal was excluded from thestatistical evaluation.

For testing SAg-glycosylceramide complexes and SAg lipopolysaccharidecomplexes, additional models are used to assess the dependence of theantitumor effect on NKT cells. Natural killer T cells (NKT) lymphocytesexpress an invariant TCR encoded by the V14 and J_(a)281 gene segments.Mice with a deletion of J_(a)281 exclusively lack V14. The V14 NKTcell-deficient mice no longer mediate IL-12 induced rejection of tumors.

Also generated are transgenic mice lacking recombination activatinggene(RAG) which preferentially generate V14 NKT cells but block thedevelopment of other lymphocyte lineages, including NK, B, and T cells.These mice are termed V14 NKT mice. J281+/+(wild type), J281−/−(deletedof V14) and RAG−/−V14tgV8.2tg (deleted of NK, T and B cells butpreferentially generate V14 NKT cells) mice are injected

-   (a) with 2×10⁶ B16 or FBL-3 (erythroleukemia) cells in the spleen to    induce liver metastasis,-   (b) intravenously with 3×10⁵ B16 or 2×10⁶ LLC (Lewis lung carcinoma)    cells for pulmonary metastases or-   (c) subcutaneously with 2×10⁶ B16 cells (melanoma) for subcutaneous    tumor growth on day 0.

SAg conjugates or fusion proteins are injected in doses of 0.1 to 50 mgon days 3, 5, 7, and 9 after the day of tumor implantation. Controlanimals are injected with PBS on the same schedule. On day 14, the miceare killed and either metastatic nodules counted or GM3 melanomaantigens measured by radioimmunoassay as previously described. Forsubcutaneous tumor growth, injection of IL-12 or PBS is initiated on day5, and the mice are treated five times per week. The diameters of tumorsare measured daily with calipers. The sizes of the tumor are expressedas the products of the longest diameter times the shortest diameter (inmm²).

EXAMPLE 17 Preparation and Administration of DNA Liposome Complexes

A representative protocol for administration of DNA-liposome complexesis as follows: DNA liposome complexes are mixed immediately prior toinjection by adding 0.1 ml of lactated Ringer's solution into a sterilevial of plasmid DNA (20 mg/ml; 0.1 ml). An aliquot of this solution (0.1ml) is added at room temperature to 0.1 ml of 150 mM (dioleoylphosphatidylethanolamine/3□[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol) liposome in lactated Ringer's solution in a separatesterile vial. The DNA and liposome vials are prepared in accordance withFDA guidelines and quality control procedures. After incubation for 15minutes at room temperature, an additional 0.5 ml of sterile lactatedRinger's solution is added to the vial and mixed. The DNA liposomesolution (0.2 ml) is injected into the patient's tumor nodule understerile conditions at the bedside after administration of localanesthesia (1% lidocaine) using a 22-gauge needle. For catheterdelivery, the DNA liposome solution (0.6 ml) is delivered into theartery using percutaneous delivery. Additional protocols foradministration of DNA liposomal constructs are given in Nabel, G J,Methods for Liposome-Mediated Gene Transfer to Tumor Cells in vivo, in:Methods in Molecular Medicine, Gene Therapy Protocols, Robbins P ed.Humana Press, Totowa N.J. (1996). Cationic liposomes for delivery of DNAconstruct to the tumor endothelium are prepared by the method ofThurston et al., J. Clin Invest., 101: 1401-1413, (1998).

EXAMPLE 18 General Procedures for Administering Constructs in HumanTumor Models and Human Patients

The constructs described herein are tested for therapeutic efficacy inseveral well established rodent models which are considered to be highlyrepresentative as described in “Protocols for Screening Chemical Agentsand Natural Products Against Animal Tumors and Other Biological Systems(Third Edition)”, Cancer Chemother. Reports, Part 3, 3: 1-112, which ishereby incorporated by reference in its entirety. Additional tumormodels of carcinoma and sarcoma originating from primary sites andprepared as established tumors at primary and/or metastatic sites areutilized to test further the efficacy of the constructs.

EXAMPLE 19 General Procedures for Administering Tumor Cells or SickledErythrocytes Transduced with SAgs and SAg-Activated T or NKT Cells inHuman Tumor Models and Human Patients

A. Tumor Cells Transduced with SAg Nucleic Acids Alone or Cotransfectedwith Oncogenes or Nucleic Acids Encoding Potent Immunogens and BacterialProducts

In a representative protocol, using the B16 melanoma or A20 lymphoma orother models given above, 10⁵-10⁷ transfected tumor cells are implantedsubcutaneously and 1-6 months later 10⁵-10⁷ untransfected tumor cells,are implanted. In the case of tumor cells cotransfected with severaltherapeutic nucleic acids, controls are established consisting of groupstransfected with only one of the nucleic acids. These singletransfectants are administered on the same schedule as thecotransfectants and assessed for capacity to prevent or reverse tumorgrowth compared to positive controls receiving tumor alone. The animalsreceiving the SAg transfected tumor cells show no evidence of growth ofthe wild type tumor and prolonged survival compared to the controls inwhich there is 100% appearance of the tumors. The differences arestatistically significant. SAg transfected tumor cells are also used totreat established tumors as follows. Transfected tumor cells, 10⁵-10⁷are given 3-10 days after the appearance of established tumors. Resultsshow statistically significant arrest of tumor growth, prolongation ofsurvival in treated animals compared to untreated controls.

B. SAg Activated Effector T or NKT Cells

Effector T or NKT cells are generated as described elsewhere and areinfused intravenously in doses of 10⁶-10⁸ into syngeneic hosts that havepulmonary metastatic lesions established by injecting tumor cellsintravenously 3 to 12 days earlier. Twenty days later, the animals aresacrificed and pulmonary metastases measured in treated animals comparedto untreated controls. Results show statistically significant reductionin total number of pulmonary nodules and prolonged survival in thetreated group compared to untreated controls.

EXAMPLE 20 General Test Evaluation Procedures for Constructs and SAgActivated Effector T or NKT Cells

I. General Test Evaluation Procedures

A. Calculation of Mean Survival Time

Mean survival time is calculated according to the following formula:${{Mean}\quad{survival}\quad{time}\quad({days})} = \frac{S + {AS}_{({A - 1})} - {\left( {B + 1} \right){NT}}}{S_{({A - 1})} - {NT}}$Definitions:

-   Day: Day on which deaths are no longer considered due to drug    toxicity. Example:

with treatment starting on Day 1 for survival systems (such as L1210,P388, B16, 3LL, and W256):

-   Day A: Day 6.-   Day B: Day beyond which control group survivors are considered    “no-takes.”-   Example: with treatment starting on Day 1 for survival systems (such    as L1210, P388, and W256), Day B−Day 18. For B16, transplanted AKR,    and 3LL survival systems, Day B is to be established.-   S: If there are “no-takes” in the treated group, S is the sum from    Day A through Day B. If there are no “no-takes” in the treated    group, S is the sum of daily survivors from Day A onward.-   S_((A−1)): Number of survivors at the end of Day (A-1). Example: for    3LE21, S_((A−1))=number of survivors on Day 5.-   NT: Number of “no-takes” according to the criteria given in    Protocols 7.300 and 11.103.    B. T/C Computed for all Treated Groups

T/C is the ratio (expressed as a percent) of the mean survival time ofthe treated group divided by the mean survival time of the controlgroup. Treated group animals surviving beyond Day B, according to thechart below, are eliminated from calculations: No. of survivors inPercent of “no-takes” treated group beyond Day B in control groupConclusion 1 Any percent “no-take” 2 <10 drug inhibition ≧10 “no-takes”≧3 <15 drug inhibitions ≧15 “no-takes”Positive control compounds are not considered to have “no-takes”regardless of the number of “no-takes” in the control group. Thus, allsurvivors on Day B are used in the calculation of T/C for the positivecontrol. Surviving animals are evaluated and recorded on the day ofevaluation as# “cures” or “no-takes.”Calculation of Median Survival Time

Median Survival Time is defmed as the median day of death for a test orcontrol group. If deaths are arranged in chronological order ofoccurrence (assigning to survivors, on the final day of observation, a“day of death” equal to that day), the median day of death is a dayselected so that one half of the animals died earlier and the other halfdied later or survived. If the total number of animals is odd, themedian day of death is the day that the middle animal in thechronological arrangement died. If the total number of animals is even,the median is the arithmetical mean of the two middle values. Mediansurvival time is computed on the basis of the entire population andthere are no deletion of early deaths or survivors, with the followingexception:

C. Computation of Median Survival Time From Survivors

If the total number of animals including survivors (N) is even, themedian survival time (days) (X+Y)/2, where X is the earlier day when thenumber of survivors is N/2, and Y is the earliest day when the number ofsurvivors (N/2)−1. If N is odd, the median survival time (days) is X.

D. Computation of Median Survival Time From Mortality Distribution

If the total number of animals including survivors (N) is even, themedian survival time (days) (X+Y)/2, where X is the earliest day whenthe cumulative number of deaths is N/2, and Y is the earliest day whenthe cumulative number of deaths is (N/2)+1. If N is odd, the mediansurvival time (days) is X.

Cures and “No-Takes”: “Cures” and “no-takes” in systems evaluated bymedian survival time are based upon the day of evaluation. On the day ofevaluation any survivor not considered a “no-take” is recorded as a“cure.” Survivors on day of evaluation are recorded as “cures” or“no-takes,” but not eliminated from the calculation of the mediansurvival time.

E. Calculation of Approximate Tumor Weight From Measurement of TumorDiameters with Vernier Calipers

The use of diameter measurements (with Vernier calipers) for estimatingtreatment effectiveness on local tumor size permits retention of theanimals for lifespan observations. When the tumor is implanted sc, tumorweight is estimated from tumor diameter measurements as follows. Theresultant local tumor is considered a prolate ellipsoid with one longaxis and two short axes. The two short axes are assumed to be equal. Thelongest diameter (length) and the shortest diameter (width) are measuredwith Vernier calipers. Assuming specific gravity is approximately 1.0,and Pi is about 3, the mass (in mg) is calculated by multiplying thelength of the tumor by the width squared and dividing the product bytwo. Thus,${{Tumor}\quad{weight}\quad({mg})} = {\frac{{length}\quad({mm}) \times \left( {{width}\quad\lbrack{mm}\rbrack} \right)^{2}}{2}\quad{Or}\quad\frac{L \times (W)^{2}}{2}}$

The reporting of tumor weights calculated in this way is acceptableinasmuch as the assumptions result in as much accuracy as theexperimental method warrants.

F. Calculation of Tumor Diameters

The effects of a drug on the local tumor diameter may be reporteddirectly as tumor diameters without conversion to tumor weight. Toassess tumor inhibition by comparing the tumor diameters of treatedanimals with the tumor diameters of control animals, the three diametersof a tumor are averaged (the long axis and the two short axes). A tumordiameter T/C of 75% or less indicates activity and a T/C of 75% isapproximately equivalent to a tumor weight T/C of 42%.

G. Calculation of Mean Tumor Weight from Individual Excised Tumors

The mean tumor weight is defmed as the sum of the weights of individualexcised tumors divided by the number of tumors. This calculation ismodified according to the rules listed below regarding “no-takes.” Smalltumors weighing 39 mg or less in control mice or 99 mg or less incontrol rats, are regarded as “no-takes” and eliminated from thecomputations. In treated groups, such tumors are defined as “no-takes”or as true drug inhibitions according to the following rules: Percent ofsmall tumors Percent of “no-takes” in treated group in control groupAction ≦17 Any percent no-take; not used in calculations 18-39 <10 druginhibition; use in calculations ≧10 no-takes; not used in calculations≧40 <15 drug inhibition; use in calculations ≧15 Code all nontoxic tests“33”

Positive control compounds are not considered to have “no-takes”regardless of the number of “no-takes” in the control group. Thus, thetumor weights of all surviving animals are used in the calculation ofT/C for the positive control. T/C are computed for all treated groupshaving more than 65% survivors. The T/C is the ratio (expressed as apercent) of the mean tumor weight for treated animals divided by themean tumor weight for control animals. SDs of the mean control tumorweight are computed the factors in a table designed to estimate SD usingthe estimating factor for SD given the range (difference between highestand lowest observation). Biometrik Tables for Statisticians (Pearson ES, and Hartley H G, eds.) Cambridge Press, vol. 1, table 22, p. 165.

II. Specific Tumor Models

A. Lymphoid Leukemia L1210

-   Summary: Ascitic fluid from donor mouse is transferred into    recipient BDF₁ or CDF₁ mice. Treatment begins 24 hours after    implant. Results are expressed as a percentage of control survival    time. Under normal conditions, the inoculum site for primary    screening is i.p., the composition being tested is administered    i.p., and the parameter is mean survival time. Origin of tumor line:    induced in 1948 in spleen and lymph nodes of mice by painting skin    with MCA. J Natl Cancer Inst. 13:1328, 1953.    Animals-   Propagation: DBA/2 mice (or BDF₁ or CDF₁ for one generation).-   Testing: BDF₁ (C57BL/6×DBA/2) or CDF₁ (BALB/c×DBA/2) mice.-   Weight: Within a 3-g weight range, with a minimum weight of 18 g for    males and 17 g for females.-   Sex: One sex used for all test and control animals in one    experiment.-   Experiment Size: Six animals per test group.-   Control Groups: Number of animals varies according to number of test    groups.    Tumor Transfer-   Inject i.p., 0.1 ml of diluted ascitic fluid containing 10⁵ cells.-   Time of Transfer for Propagation: Day 6 or 7.-   Time of Transfer for Testing: Day 6 or 7.    Testing Schedule-   Day 0: Implant tumor. Prepare materials. Run positive control in    every odd-numbered experiment. Record survivors daily.-   Day 1: Weigh and randomize animals. Begin treatment with therapeutic    composition. Typically, mice receive 1 ug of the test composition in    0.5 ml saline. Controls receive saline alone. The treatment is given    as one dose per week. Any surviving mice are sacrificed after 4    weeks of therapy.-   Day 5: Weigh animals and record.-   Day 20: If there are no survivors except those treated with positive    control compound, evaluate study.-   Day 30: Kill all survivors and evaluate experiment.    Quality Control

Acceptable control survival time is 8-10 days. Positive control compoundis 5-fluorouracil; single dose is 200 mg/kg/injection, intermittent doseis 60 mg/kg/injection, and chronic dose is 20 mg/kg/injection. Ratio oftumor to control (T/C) lower limit for positive control compound is 135%

Evaluation

Compute mean animal weight on Days 1 and 5, and at the completion oftesting compute T/C for all test groups with >65% survivors on Day 5. AT/C value 85% indicates a toxic test. An initial T/C 125% is considerednecessary to demonstrate activity. A reproduced T/C 125% is consideredworthy of further study. For confirmed activity a composition shouldhave two multi-dose assays that produce a T/C 125%.

B. Lymphocytic Leukemia P388

-   Summary: Ascitic fluid from donor mouse is implanted in recipient    BDF₁ or CDF₁ mice. Treatment begins 24 hours after implant. Results    are expressed as a percentage of control survival time. Under normal    conditions, the inoculum site for primary screening is ip, the    composition being tested is administered ip daily for 9 days, and    the parameter is median survival time. Origin of tumor line: induced    in 1955 in a DBA/2 mouse by painting with MCA. Scientific    Proceedings, Pathologists and Bacteriologists 33:603, 1957.    Animals-   Propagation: DBA/2 mice (or BDF₁ or CDF₁ for one generation)-   Testing: BDF₁ (C57BL/6×DBA/2) or CDF₁ (BALB/c×DBA/2) mice.-   Weight: Within a 3-g weight range, with a minimum weight of 18 g for    males and 17 g for females.-   Sex: One sex used for all test and control animals in one    experiment.-   Experiment Size: Six animals per test group.-   Control Groups: Number of animals varies according to number of test    groups.    Tumor Transfer-   Implant: Inject ip-   Size of Implant: 0.1 ml diluted ascitic fluid containing 10⁶ cells.-   Time of Transfer for Propagation: Day 7.-   Time of Transfer for Testing: Day 6 or 7.    Testing Schedule-   Day 0: Implant tumor. Prepare materials. Run positive control in    every odd-numbered experiment. Record survivors daily.-   Day 1: Weigh and randomize animals. Begin treatment with therapeutic    composition. Typically, mice receive lug of the composition being    tested in 0.5 ml saline. Controls receive saline alone. The    treatment is given as one dose per week. Any surviving mice are    sacrificed after 4 weeks of therapy.-   Day 5: Weigh animals and record.-   Day 20: If there are no survivors except those treated with positive    control compound, evaluate experiment.-   Day 30: Kill all survivors and evaluate experiment.    Quality Control

Acceptable median survival time is 9-14 days. Positive control compoundis 5-fluorouracil: single dose is 200 mg/kg/injection, intermittent doseis 60 mg/kg/injection, and chronic dose is 20 mg/kg/injection. T/C lowerlimit for positive control compound is 135% Check control deaths, notakes, etc.

Evaluation

Compute mean animal weight on Days 1 and 5, and at the completion oftesting compute T/C for all test groups with >65% survivors on Day 5. AT/C value 85% indicates a toxic test. An initial T/C 125% is considerednecessary to demonstrate activity. A reproduced T/C 125% is consideredworthy of further study. For confirmed activity a synthetic must havetwo multi-dose assays (each performed at a different laboratory) thatproduce a T/C 125%; a natural product must have two different samplesthat produce a T/C 125% in multi-dose assays.

C. Melanotic Melanoma B16

-   Summary: Tumor homogenate is implanted ip or sc in BDF₁ mice.    Treatment begins 24 hours after either ip or sc implant or is    delayed until an sc tumor of specified size (usually approximately    400 mg) can be palpated. Results expressed as a percentage of    control survival time. The composition being tested is administered    ip, and the parameter is mean survival time. Origin of tumor line:    arose spontaneously in 1954 on the skin at the base of the ear in a    C57BL/6 mouse. Handbook on Genetically Standardized Jax Mice.    Roscoe B. Jackson Memorial Laboratory, Bar Harbor, Maine, 1962. See    also Ann NY Acad Sci 100, Parts 1 and 2, 1963.    Animals-   Propagation: C57BL/6 mice.-   Testing: BDF₁ (C57BL/6×DBA/2) mice.-   Weight: Within a 3-g weight range, with a minimum weight of 18 g for    males and 17 g for females.-   Sex: One sex used for all test and control animals in one    experiment.-   Experiment Size: Ten animals per test group. For control groups, the    number of animals varies according to number of test groups.    Tumor Transfer-   Propagation: Implant fragment sc by trochar or 12-gauge needle or    tumor homogenate (see below) every 10-14 days into axillary region    with puncture in inguinal region. Testing: Excise sc tumor on Day    10-14.-   Homogenate: Mix 1 g or tumor with 10 ml of cold balanced salt    solution and homogenize, and implant 0.5 ml of this tumor homogenate    ip or sc.-   Fragment: A 25-mg fragment may be implanted sc.    Testing Schedule-   Day 0: Implant tumor. Prepare materials. Run positive control in    every odd-numbered experiment. Record survivors daily.-   Day 1: Weigh and randomize animals. Begin treatment with therapeutic    composition. Typically, mice receive 1 μg of the composition being    tested in 0.5 ml saline. Controls receive saline alone. The    treatment is given as one dose per week. Any surviving mice are    sacrificed 8 weeks of therapy.-   Day 5: Weigh animals and record.-   Day 60: Kill all survivors and evaluate experiment.    Quality Control

Acceptable control survival time is 14-22 days. Positive controlcompound is 5-fluorouracil: single dose is 200 mg/kg/injection,intermittent dose is 60 mg/kg/injection, and chronic dose is 20mg/kg/injection. T/C lower limit for positive control compound is 135%Check control deaths, no takes, etc.

Evaluation

Compute mean animal weight on Days 1 and 5, and at the completion oftesting compute T/C for all test groups with >65% survivors on Day 5. AT/C value 85% indicates a toxic test. An initial T/C 125% is considerednecessary to demonstrate activity. A reproduced T/C 125% is consideredworthy of further study. For confirmed activity a therapeuticcomposition should have two multi-dose assays that produce a T/C 125%.

Metastasis after IV Injection of Tumor Cells

10⁵ B16 melanoma cells in 0.3 ml saline are injected intravenously inC57BL/6 mice. The mice are treated intravenously with Ig of thecomposition being tested in 0.5 ml saline. Controls receive salinealone. The treatment is given as one dose per week. Mice sacrificedafter 4 weeks of therapy, the lungs are removed and metastases areenumerated.

C. 3LL Lewis Lung Carcinoma

-   Summary: Tumor may be implanted sc as a 2-4 mm fragment, or im as a    2×10⁶-cell inoculum. Treatment begins 24 hours after implant or is    delayed until a tumor of specified size (usually approximately 400    mg) can be palpated. The composition being tested is administered ip    daily for 11 days and the results are expressed as a percentage of    the control.-   Origin of tumor line: arose spontaneously in 1951 as carcinoma of    the lung in a C57BL/6 mouse. Cancer Res 15:39, 1955. See, also    Malave, I. et al., J. Nat'l. Canc. Inst. 62:83-88 (1979).    Animals-   Propagation: C57BL/6 mice.-   Testing: BDF₁ mice or C3H.-   Weight: Within a 3-g weight range, with a minimum weight of 18 g for    males and 17 g for females.-   Sex: One sex used for all test and control animals in one    experiment.-   Experiment Size: Six animals per test group for sc implant, or ten    for im implant. For control groups, the number of animals varies    according to number of test groups.    Tumor Transfer-   Implant: Inject cells im in hind leg or implant fragment sc in    axillary region with puncture in inguinal region.-   Time of Transfer for Propagation: Days 12-14.-   Time of Transfer for Testing: Days 12-14.    Testing Schedule-   Day 0: Implant tumor. Prepare materials. Run positive control in    every odd-numbered experiment. Record survivors daily.-   Day 1: Weigh and randomize animals. Begin treatment with therapeutic    composition. Typically, mice receive lug of the composition being    tested in 0.5 ml saline. Controls receive saline alone. The    treatment is given as one dose per week. Any surviving mice are    sacrificed after 4 weeks of therapy.-   Day 5: Weigh animals and record.-   Final Day: Kill all survivors and evaluate experiment.    Quality Control

Acceptable im tumor weight on Day 12 is 500-2500 mg. Acceptable im tumormedian survival time is 18-28 days. Positive control compound iscyclophosphamide: 20 mg/kg/injection, qd, Days 1-11. Check controldeaths, no takes, etc.

Evaluation

Compute mean animal weight when appropriate, and at the completion oftesting compute T/C for all test groups. When the parameter is tumorweight, a reproducible T/C 42% is considered necessary to demonstrateactivity. When the parameter is survival time, a reproducible T/C 125%is considered necessary to demonstrate activity. For confirmed activitya synthetic must have two multi-dose assays (each performed at adifferent laboratory); a natural product must have two differentsamples.

D. 3LL Lewis Lung Carcinoma Metastasis Model

This model has been utilized by a number of investigators. See, forexample, Gorelik, E. et al., J. Nat'l. Canc. Inst. 65:1257-1264 (1980);Gorelik, E. et al., Rec. Results Canc. Res. 75:20-28 (1980); Isakov, N.et al., Invasion Metas. 2:12-32 (1982) Talmadge J. E. et al., J. Nat'l.Canc. Inst. 69:975-980 (1982); Hilgard, P. et al., Br. J. Cancer35:78-86(1977)).

-   Mice: male C57BL/6 mice, 2-3 months old.-   Tumor: The 3LL Lewis Lung Carcinoma was maintained by sc transfers    in C57BL/6 mice. Following sc, im or intra-footpad transplantation,    this tumor produces metastases, preferentially in the lungs.    Single-cell suspensions are prepared from solid tumors by treating    minced tumor tissue with a solution of 0.3% trypsin. Cells are    washed 3 times with PBS (pH 7.4) and suspended in PBS. Viability of    the 3LL cells prepared in this way is generally about 95-99% (by    trypan blue dye exclusion). Viable tumor cells (3×10⁴-5×10⁶ )    suspended in 0.05 ml PBS are injected into the right hind foot pads    of C57BL/6 mice. The day of tumor appearance and the diameters of    established tumors are measured by caliper every two days.

Typically, mice receive lug of the composition being tested in 0.5 mlsaline.

Controls receive saline alone. The treatment is given as one or twodoses per week.

In experiments involving tumor excision, mice with tumors 8-10 mm indiameter are divided into two groups. In one group, legs with tumors areamputated after ligation above the knee joints. Mice in the second groupare left intact as nonamputated tumor-bearing controls. Amputation of atumor-free leg in a tumor-bearing mouse has no known effect onsubsequent metastasis, ruling out possible effects of anesthesia, stressor surgery. Surgery is performed under Nembutal anesthesia (60 mgveterinary Nembutal per kg body weight).

Determination of Metastasis Spread and Growth

Mice are killed 10-14 days after amputation. Lungs are removed andweighed. Lungs are fixed in Bouin's solution and the number of visiblemetastases is recorded. The diameters of the metastases are alsomeasured using a binocular stereoscope equipped with amicrometer-containing ocular. under 8× magnification. On the basis ofthe recorded diameters, it is possible to calculate the volume of eachmetastasis. To determine the total volume of metastases per lung, themean number of visible metastases is multiplied by the mean volume ofmetastases. To further determine metastatic growth, it is possible tomeasure incorporation of ¹²⁵IdUrd into lung cells (Thakur, M. L. et al.,J. Lab. Clin. Med. 89:217-228 (1977). Ten days following tumoramputation, 25 μg of FdUrd is inoculated into the peritoneums oftumor-bearing (and, if used, tumor-resected mice. After 30 min, mice aregiven 1 μCi of ¹²⁵IdUrd. One day later, lungs and spleens are removedand weighed, and a degree of ¹²⁵IdUrd incorporation is measured using agamma counter.

Statistics: Values representing the incidence of metastases and theirgrowth in the lungs of tumor-bearing mice are not normally distributed.Therefore, non-parametric statistics such as the Mann-Whitney U-Test maybe used for analysis. Study of this model by Gorelik et al. (1980,supra) showed that the size of the tumor cell inoculum determined theextent of metastatic growth. The rate of metastasis in the lungs ofoperated mice was different from primary tumor-bearing mice. Thus in thelungs of mice in which the primary tumor had been induced by inoculationof large doses of 3LL cells (1-5×10⁶) followed by surgical removal, thenumber of metastases was lower than that in nonoperated tumor-bearingmice, though the volume of metastases was higher than in the nonoperatedcontrols. Using ¹²⁵IdUrd incorporation as a measure of lung metastasis,no significant differences were found between the lungs of tumor-excisedmice and tumor-bearing mice originally inoculated with 1×10⁶ 3LL cells.Amputation of tumors produced following inoculation of 1×10⁵ tumor cellsdramatically accelerated metastatic growth. These results were in accordwith the survival of mice after excision of local tumors. The phenomenonof acceleration of metastatic growth following excision of local tumorshad been observed by other investigators. The growth rate and incidenceof pulmonary metastasis were highest in mice inoculated with the lowestdoses (3×10 ⁴-1×10⁵ of tumor cells) and characterized also by thelongest latency periods before local tumor appearance. Immunosuppressionaccelerated metastatic growth, though nonimmunologic mechanismsparticipate in the control exerted by the local tumor on lung metastasisdevelopment. These observations have implications for the prognosis ofpatients who undergo cancer surgery.

E. Walker Carcinosarcoma 256

-   Summary: Tumor may be implanted sc in the axillary region as a 2-6    mm fragment, im in the thigh as a 0.2-ml inoculum of tumor    homogenate containing 10⁶ viable cells, or ip as a 0.1-ml suspension    containing 10⁶ viable cells. Treatment of the composition being    tested is usually ip. Origin of tumor line: arose spontaneously in    1928 in the region of the mammary gland of a pregnant albino rat. J    Natl Cancer Inst 13:1356, 1953.    Animals-   Propagation: Random-bred albino Sprague-Dawley rats.-   Testing: Fischer 344 rats or random-bred albino rats.-   Weight Range: 50-70 g (maximum of 10-g weight range within each    experiment).-   Sex: One sex used for all test and control animals in one    experiment.-   Experiment Size: Six animals per test group. For control groups, the    number of animals varies according to number of test groups.    Time of Tumor Transfer-   Time of Transfer for Propagation: Day 7 for im or ip implant; Days    11-13 for sc implant.-   Time of Transfer for Testing: Day 7 for im or ip implant; Days 11-13    for sc implant.    Tumor Transfer

Sc fragment implant is by trochar or 12-gauge needle into axillaryregion with puncture in inguinal area. Im implant is with 0.2 ml oftumor homogenate (containing 10⁶ viable cells) into the thigh. Ipimplant is with 0.1 ml of suspension (containing 10⁶ viable cells) intothe ip cavity.

Testing Schedule

Prepare and administer compositions under test on days, weigh animals,and evaluate test on the days listed in the following tables. Testsystem Prepare drug Administer drug Weight animals Evaluate 5WA16 2 3-63 and 7  7 5WA12 0 1-5 1 and 5 10-14 5WA31 0 1-9 1 and 5 30

-   Day 0: Implant tumor. Prepare materials. Run positive control in    every odd-numbered experiment. Record survivors daily.-   Day 1: Weigh and randomize animals.-   Final Day: Kill all survivors and evaluate experiment.    Quality Control

Acceptable im tumor weight or survival time for the above three testsystems: 5WA16: 3-12 g. 5WA12: 3-12 g. 5WA31 or 5WA21: 5-9 days.

Evaluation

Compute mean animal weight when appropriate, and at the completion oftesting compute T/C for all test groups. When the parameter is tumorweight, a reproducible T/C 42% is considered necessary to demonstrateactivity. When the parameter is survival time, a reproducible T/C 125%is considered necessary to demonstrate activity. For confirmed activitya therapeutic agent must have activity in two multi-dose assays.

F. A20 lymphoma

10⁶ murine A20 lymphoma cells in 0.3 ml saline are injectedsubcutaneously in Balb/c mice. The mice are treated intravenously with 1g of the composition being tested in 0.5 ml saline. Controls receivesaline alone. The treatment is given as one dose per week. Tumor growthis monitored daily by physical measurement of tumor size and calculationof total tumor volume. After 4 weeks of therapy the mice are sacrificed.

Treatment Regimens and Results (Constructs)

For determining efficacy in the tumor models described above the generalcategories of therapeutic constructs used are given below. For all ofthe classes of conjugates listed below, the SAg component can beprepared as either a DNA encoding SAg or as the SAg polypeptide itself.In either form the SAg DNA or protein may be conjugated to additionalmolecules, either nucleic acid or polypeptides. Operationally, fortherapeutic use in vivo or ex vivo, these conjugates may be prepared bychemical coupling or by recombinant means (whichever is appropriate) andconjugated to a tumor-targeting structure or incorporated into a vehicle(e.g., liposomes) that themselves comprise a tumor targetingstructure(s). Again, examples of such targeting structures include, butare not limited to, an antibody, antigen, receptor or receptor ligand.Methods are disclosed in Examples 1, 3, 4, 5, 6, 7, 14, 17, 18, 30-32.

-   1. SAg Nucleic Acid Constructs including Phage Displays and SAg    Transfected Bacterial Cells-   2. Glycosylated SAgs-   3. Chimeric SAgs

Conjugates having a Superantigen component (polypeptide or nucleic acid)and a partner that is either a single component or a conjugate of 2 ormore components (protein, carbohydrate, lipid or DNA) as indicatedbelow. Superantigen (Protein or DNA) Partner (Single Component orConjugate) 4. DNA coding sequence 5. Polypeptide 6. Nucleic acid 7.Tumor associated Peptide 8. Tumor Antigen-MHC protein 9. LPS 10.Lipoarabinomannan 11. Ganglioside 12. Glycosphingolipid 13.Ganglioside-CD1 receptor 14. Glycosphingolipid-CD1 receptor 15.Glycosylceramide (e.g., Gal-Cer) 16. GalCer-CD1 receptor 17. Gal 18.Arg-Gly-Asp or Asn-Gly-Arg 19 iNOS 20. Gb2 or Gb3 or Gb4 18. (Gb2 or Gb3or Gb4)-CD1 receptor   19. -GPI-(Gb2 or Gb3 or Gb4)   21. -GPI-(Gb2 orGb3 or Gb4)-CD1 receptor    22. Verotoxin 23. Verotoxin A or BSubunit     24. IFNα receptor peptide homologous to VT 25. CD19 peptidehomologous to VT 26. LDL, VLDL, HDL, IDL 27. Apolipoproteins (e.g.,Lp(a), apoB-100, apoB-48, apoE) 28. OxyLDL, oxyLDL mimics, (e.g., 7□-hydroperoxycholesterol, 7□-hydroxycholesterol, 7-ketocholesterol,5α-6α-epoxycholesterol, 7□-hydroperoxy-choles-5-en-3□-ol,4-hydroxynonenal (4-HNE), 9-HODE, 13-HODE and cholesterol-9-HODE) 29.OxyLDL by products (e.g. lysolecithin, lysophosphatidylcholine,malondialdehyde, 4-hydroxynonenal) 30 LDL & oxyLDL receptors (e.g., LDLoxyLDL, acetyl-LDL, VLDL, LRP, CD36, SREC, LOX-1, macrophage scavengerreceptors)Vaccine Use

For use as a vaccine, the constructs are administered subcutaneously,intramuscularly intradermally or intraperitoneally in doses ranging from50 to 500 ng in various vehicles such as Freund's adjuvant, aluminumhydroxide, pluruonic acid triblock and liposomes as described in theart. Doses may be repeated every 10 days. Tumors are implanted after thelast dose. A control group does not receive the vaccine.

Use in Established Tumors

For proteins or nucleic acid constructs, treatment consists of injectinganimals iv or ip with 50, 500 1000 or 5,000 ng of in 0.1-0.5 ml ofnormal saline. Unless indicated otherwise above, treatments are givenone to three times per week for two to five weeks. Phage displays areadministered as 10⁹ transducing units (TU) and irradiated bacterialcells as 10⁵ cells iv into the tail vein one to three times per week fortwo to five weeks. Exosomes or vesicles, harvested from transfected,transformed or fusion tumor cells or sickled cells are given i.v. intothe tail vein in a dose of 0.25-1 g per animal one to three times perweek for two to five weeks. The results shown in Table VI are for eachcomposition and dose tested. The results are statistically significantby the Wilcoxon rank sum test.

Treatment regimens for SAg activated effector T or NKT cells are inExample 16, 18, 19. The preferred animal model for evaluation of theadoptively transferred T or NKT effector cells is the MCA 205/207fibrosarcoma with pulmonary metastases (Shu S. et al., J. Immunol. 152:1277-1288 (1994)). The other models given in Example 20 are alsosuitable for evaluation of the therapeutic effectiveness of the effectorT cells. TABLE VI Tumor Model Parameter % of Control Response L1210 Meansurvival time >130% P388 Mean survival time >130% B16 Mean survivaltime >130% B16 metastasis Median number of metastases <70% 3LL Meansurvival time >130% Mean tumor weight <40% 3LL metastasis Mediansurvival time >130% Mean lung weight <60 Median number of metastases<60% Median volume of metastases <60% Medial volume of metastases <60%Median uptake of IdUrd <60% Walker carcinoma Median survival time >130%Mean tumor weight <40% A20 Mean survival time >130% Mean tumor volume<40%

EXAMPLE 22 Antitumor Effects of Therapeutic Constructs and Effector T,NKT Cells or Sickled Erythrocytes in Human Patients

All patients treated have histologically confirmed malignant diseaseincluding carcinomas, sarcomas, melanomas, lymphomas and leukemia andhave failed conventional therapy. Patients may be diagnosed as havingany stage of metastatic disease involving any organ system. Stagingdescribes both tumor and host, including organ of origin of the tumor,histologic type and histologic grade, extent of tumor size, site ofmetastases and functional status of the patient. A generalclassification includes the known ranges of Stage I (localized disease)to Stage 4(widespread metastases). Patient history is obtained andphysical examination performed along with conventional tests ofcardiovascular and pulmonary function and appropriate radiologicprocedures. Histopathology is obtained to verify malignant disease.

EXAMPLE 23 Treatment Procedures

Constructs (or Preparations)

Doses of the constructs are determined as described above using, interalia, appropriate animal models of tumors. Two classes of therapeuticcompositions are administered namely SAg proteins or SAg conjugates asdescribed above for animal models.

A treatment consists of injecting the patient with 0.5-500 mg ofConstruct intravenously in 200 ml of normal saline over a one hourperiod. Treatments are given 3×/week for a total of 12 treatments.Patients with stable or regressing disease are treated beyond the12^(th) treatment. Treatment is given on either an outpatient orinpatient basis as needed.

Effector T or NKT Cells

Eligible patients are treated with tumor antigens such as irradiatedtumor cells or GM-CSF transduced tumor cells injected approximately 10centimeters from a draining lymph node site. Ten days post injection,draining lymph nodes are obtained in a limited surgical procedure at thesite draining the injection. The lymph nodes are converted to a singlecell suspension of lymphocytes and these are incubated with various SAgpreparations for two days followed by Il-2 for an additional 72 hours.These lymphocytes now called effector T cells or NKT cell are used foradoptive immunotherapy.

Effector T or NKT cells harvested by centrifugation at 500×g for 15 minand the cell pellets are pooled. After washing the cells in HBSS, thecell are resuspended in 200 ml of normal saline containing 5% humanserum albumin and 450,000 IU of IL-2 for transfer. Each recipient willreceive four escalating doses or 33 million, 100 million, 330 millionand 1 billion cells per square meter of body surface area each given oneweek apart. Cells are infused through a subclavian central venouscatheter over a 30-minute interval. IL-2 administration i.v. iscommenced immediately after completion of cell infusion at a dose andschedule of 180,000 IU/ml every 8 h. for 5 days. All patients receiveindomethacin (50 mg P.O.) every 8 h, acetaminophen (650 mg P.O.) every 6h. and ranitidine (150 mg P.O.) every 12 h while receiving IL-2 in orderto reduce febrile and gastric side effects. As controls, a cohort ofpatients is treated with the in vivo tumor vaccination step and IL-2without the tumor effector cells. Patients will be followed for clinicalresponse every 4 weeks for 2 months with repeat radiologicalexaminations.

Abbreviated Exemplary Human Protocol: Sequential Administration ofGM-CSF Transduced Tumor Cells In vivo and SAg Activated NKT and T Cellsex vivo in Patients with Metastatic Renal Cell Carcinoma and Melanoma

In vivo Phase: Immunization with GM-CSF Transduced Tumor Cells

-   Day 1: GM CSF transfected tumor cells (renal carcinoma/melanoma) are    injected as given in Phase I GM-CSF Gene Transduction Protocol    [Human Gene Therapy 6: 347-368, (1995)]

-   Day 7-10: Lymph Nodes draining the GM-CSF transfected tumor cell    sites are removed and placed in tissue culture OR patients are    pheresed and their peripheral blood T cells and NKT cells collected    for further treatment in tissue culture as described below.    Ex vivo Phase: Immunization with SAg

-   1. The T cells are obtained from either lymph nodes draining GM-CSF    transduced tumor cell immunization or peripheral blood and    subdivided into CD4+CD8+ (T cell)and CD4−CD8− (NKT cell)    populations.

-   2. SAg enterotoxin B is added to cultures of the NKT and T cell    populations for 48 hours.

-   3. The NKT cells and T cells are further expanded for an additional    72 hours (optional).    SAg Activated NKT and/or T Cell Administration

-   1. The CD4+CD8+ (T cell) and CD4−CD8− (NKT) populations are    harvested for injection into patients.

-   2. T cells or NKT cells are administered with a mean 1011 cells per    patient.

-   

Assessment:

-   

1. T cells phenotypes for NKT cell markers, V expression, CD44, CD62 arecarried out on lymph node and peripheral blood T cells or NKT cellsimmediately after their removal and at various intervals after ex vivoSAg stimulation and expansion.

-   2. Tumor and DTH assessment are as described in the Phase I Protocol    on GM-CSF Transduction [Human Gene Therapy 6: 347-368 (1995)].    Patient Evaluation

Assessment of response of the tumor to the therapy is made once per weekduring therapy and 30 days thereafter. Depending on the response totreatment, side effects, and the health status of the patient, treatmentis terminated or prolonged from the standard protocol given above. Tumorresponse criteria are those established by the International UnionAgainst Cancer and are listed in Table VII. TABLE VII RESPONSEDEFINITION Complete remission Disappearance of all evidence of disease(CR) Partial remission >50% decrease in the product of the two greatest(PR) perpendicular tumor diameters; no new lesions Less than partial25-50% decrease in tumor size, stable for at least remission (<PR) 1month Stable disease <25% reduction in tumor size; no progression or newlesions Progression >25% increase in size of any one measured lesion orappearance of new lesions despite stabilization or remission of diseasein other measured sites

The efficacy of the therapy in a population is evaluated usingconventional statistical methods including, for example, the Chi Squaretest or Fisher's exact test. Long-term changes in and short term changesin measurements can be evaluated separately.

Results

One hundred and fifty patients are treated. The results are summarizedin Table VIII. Positive tumor responses are observed in 80% of thepatients as follows: TABLE VIII All Patients Response No. % PR 20 66%<PR 10 33% Tumor Types Response % of Patients Breast Adenocarcinoma PR +<PR 80% Gastrointestinal Carcinoma PR + <PR 75% Lung Carcinoma PR + <PR75% Prostate Carcinoma PR + <PR 75% Lymphoma/Leukemia PR + <PR 75% Headand Neck Cancer PR + <PR 75% Renal and Bladder Cancer PR + <PR 75%Melanoma PR + <PR 75%

EXAMPLE 24 Preparation of DCs

Splenocytes obtained from naive C57BL/6 females are treated withammonium chloride Tris buffer for 3 min at 37° C. to deplete red bloodcells. Splenocytes (3 ml) at 2×10⁷ cells/ml are layered over 2 mlmetrizamide gradient column (Nycomed Pharma AS, Oslo, Norway; analyticalgrade, 14.5 g added to 100 ml PBS, pH 7.0) and centrifuged at 600 g for10 mm. The DC-enriched fraction from the interface is further enrichedby adherence for 90 mm. Adherent cells (mostly DC and a fewcontaminating macrophages) are retrieved by gentle scraping andsubjected to a second round of adherence at 37° C. for 90 min to depletethe contaminating macrophages. Non-adherent cells are pooled as splenicDC, and by FACS® analysis are ˜80-85% DC (stainwith mAb 33D1), 1-2%macrophages (stain with mAb F4/80), 10% T cells, and <5% B cells. Thepellet is resuspended and enriched for macrophages by two rounds ofadherence at 37° C. for 90 mm each. More than 80% of the adherentpopulation is identified as macrophages by FACS® analysis with 5%lymphocytes and <5% DC. B cells are separated from the non-adherentpopulation (B and T cells) by panning on anti-Ig-coated plates. Theseparated cell population which is comprised of >80% T lymphocytes byFACS analysis is used as responder T cells

Generation of Bone Marrow-Derived DCs.

Erythrocyte depleted mouse bone marrow cells from flushed marrowcavities are cultured in CM with 10 ng/ml GM-CSF and 10 ng/ml IL-4 at1×10⁶ cells/ml. On day 7, DCs are harvested by gentle pipetting and areenriched by 14.5% (by weight) metrizamide (Sigma) CM gradients. The lowdensity interface containing the DC is collected by gentle pipetteaspiration. The floating DCs express CD11b, CD11c, CD86, DEC2O5, MHCclass I and II and CD4O. They are negative or low for CD3 and B220expression.

DC Cultures

Mouse BM-DCs are prepared in CM with IL-4 and GM-CSF (1000 IU/ml each).The DC are washed twice with CM, enumerated purity >90% by positivecoexpression of MHC class II, CD40, CD80, CD86, and CD11c byfluorescence-activated cell sorter (ACS)], and cultured in CM with addedcytokines for further studies. Human-monocyte-derived DCs are obtainedfrom the adherent fraction of mononuclear cells of healthy volunteersand are incubated 7-8 days in AIMV containing L-Glu, antibiotics andrhIL-4 and rhGM-CSF (1000 IU/ml each, Schering Plough, Kenilworth, N.J.,U.S.A). After 8 days in culture, the loosely adherent or floating cellsshow typical dendritic morphology, express high levels of MHC class Iand II molecules, CD4O and CD86; most are positive for CD1a and CD11cbut low or negative for CD2, CD3, CD14, CD19 and CD83.

EXAMPLE 25 Preparation of DC/Tumor Cells Hybrids (DC/tc)

DCs derived from BM culture are fused with tumor cells at a 3:1(DC:tumor cell) ratio using polyethylene glycol (PEG; MW=1450)/DMSOsolution (Sigma). In brief, tumor cells are cultured in CM supplementedwith 20% FCS and 1×OPI solution (oxaloacetate, pyruvate, and insulin;Sigma) for 4-6 h before fusion. Tumor cells and DCs are then mixed andwashed with serum-free medium. After removing the medium, I ml of PEG isadded to the cell pellet while resuspending the cells by stirring for 2min. An additional 10 ml of serum-free medium is added to the cellsuspension over the next 3 min. with continued stirring. The cells arecentrifuged at 400×g for 5 mm. The cells are resuspended with 20% FCS CMand cultured for 24 h before staining or being used as targets orvaccines. Fusion preparations of DCs with B16 or RMA-S are termed B16/DCand RMA-S/DC, respectively.

Phenotype Staining of Fused Hybrid Cells

B16, RMA-S, DCs, and their fused hybrids are analyzed by staining withFITC- or PE-conjugated mAbs (PharMingen) against MHC antigens (D^(b),K^(b),IA^(b)) adhesion and costimulatory molecules (B7.1, ICAM-1) andlymphocyte antigens (Thy-1.2, SmIg) at 4° C. for 45 min. DCs wereidentified by labeling with mAb against CD11c (N418). B16, B16/DC orB16/B16 fused cells are stained with mAb against AKV Env gp85 protein(M562, provided by Dr. Masaru Taniguchi, Chiba University, Tokyo, Japan)as a B16 tumor-specific marker. RMA-S and RMA-S/DC fused cells arestained with Thy-1.2 or mAb against the R-MuLV-encoded Gag p12 protein(584, provided by Dr. Bruce Chesebro, National Institute of Allergy andInfectious Diseases, Hamilton, Mo.) as RMA-S tumor-derived markers. Themethod for labeling cells with TRITC (rhodamine) is described. Briefly,cells are resuspended in RPMI 1640 at 1×10⁶ cells/ml and incubated withTRITC (0.5 g/ml) in 37° C. for 45 mm. The labeled cells are washed threetimes and used for fusion studies. The phenotypes of fresh and culturedLN T cells is determined by FACS analysis following staining with FITC-or PE-conjugated mAbs against Thy-1.2, Lyt-2, and L3T4 (PharMingen). Allcells are washed twice with HBSS and fixed with 0.2% paraformaldehyde.Fluorescence intensity and positive cell percentage were measured on aFACScan flow microfluorometer (Becton Dickinson, Sunnyvale, Calif.).

Additional Fusion Methods

Murine (CS 7BL16) MC38 adenocarcinoma cells are stably transfected withthe DF3/MUC1 cDNA (MC38/MUC1). MC38, MC38/MUC1 and the syngeneic MB49bladder cancer cells are maintained in DMEM supplemented with 10%heat-inactivated fetal calf serum (FCS), 2 mM glutamine, 100 U/mlpenicillin and 100 μg/ml streptomycin. DCs are obtained as describedfrom bone marrow culture with certain modifications. Briefly, bonemarrow is flushed from long bones, and red cells are lysed with ammoniumchloride. Lymphocytes, granulocytes and Ia⁺ cells are depleted from thebone marrow cells by incubation with the following mAbs: (1) 2.43,anti-CD8 (TIB 210; American Type Culture Collection, Rockville, Md.);(2) GK1.5, anti-CD4 (TIB 207); (3) RA3-3A1/6.1, anti B220/CD45R (TIB146); (4) B21-2, anti-Ia (TIB 229); and (5) RB6-8C5, anti-Gr-1(PharMingen, San Diego, Calif.) and then rabbit complement. The cellsare plated in six-well culture plates in RPMI 1640 medium supplementedwith 5% heat-inactivated FCS, 50 M 2-mercaptoethanol, 1 mM HEPES (pH7.4), 2 mM glutamine, 10 U/ml penicillin, 100 μg/ml streptomycin and 500U/ml recombinant murine GM-CSF (Boehringer Mannheim, Indianapolis,Ind.). At day 7 of culture, nonadherent and loosely adherent cells arecollected and replated in 100-mm petri dishes (10⁶ cells/ml; 8nil/dish). The nonadherent cells are washed away after 30 mm ofincubation, and GM-CSF in RPMI medium is added to the adherent cells.After 18 h, the nonadherent cell population is removed for fusion withMC38/MUC1 or MC38. Fusion is carried out with 50% PEG in Dulbecco's PBSwithout Ca²+ or Mg²+ at pH 7.4. The fused cells are plated in 24-wellculture plates in the presence of HAT medium (Sigma) for 10-14 days. HATslows proliferation of MC38/MUC1 and MC38, but not the fused cells.MC38/MUC1 and MC38 cells grow firmly attached to the tissue cultureflask, while the fused cells are dislodged by gentle pipetting.

Flow Cytometry

Cells are washed with PBS and incubated with mAb DF3 (anti-MUC1), mAbM1/42/3.9.8 (anti-MHC class I), mAb M5/114 (anti-MHC class II), mAb16-1OA1 (anti-B7-1), mAb GL1 (anti-B7-2) or mAb 3E, (anti-ICAM-1) for 30mm on ice. After washing with PBS, the appropriate fluoresceinisothiocyanate (FITC)-conjugated anti-hamster, -rat and -mouse IgG isadded for another 30 mm on ice. Samples are then washed, fixed andanalyzed in a FACScan (Becton Dickinson, Mountain View, Calif.).

EXAMPLE 26 Transfection of Hybrid DC/tc's with SAg DNA or RNA in vivoand in vitro

Methods of transfection of SAg-encoding nucleic acid into tumor cell aredisclosed in the Examples 1, 32. The same methods are used fortransfection of DCs or DC/tc hybrids.

EXAMPLE 27 Preparation of DCs which have Phagocytosed SAg-TransfectedTumor Cell Lysates or Apoptotic Tumor Cells

PBMCs, DCs, macrophages, and T cells are prepared as follows. In brief,peripheral blood is obtained from normal donors in heparinized syringesand PBMCs are isolated by sedimentation over Ficoll-Hypaque (AmershamPharmacia Biotech, Piscataway, N.J.). T cell-enriched and Tcell-depleted fractions are prepared by rosetting withneuraminidase-treated sheep red blood cells. Immature DCs are preparedfrom the T cell-depleted fraction by culturing cells in the presence ofGM-CSF and IL-4 for 7 d. 1,000 U/ml of GM-CSF (Immunex Corp., Seattle,Wash.) and 500-1,000 U/ml of IL-4 (Schering-Plough Corp., Kenilworth,N.J.) are added to the cultures on days 0, 2, and 4. To generate matureDCs, the cultures are transferred to fresh wells on day 7 and MCM isadded for an additional 3-4 d. At day 7, >95% of the cells areCD14−,CD83−, HLA-DR^(lo) DCs. On days 10-11, 80-100% of the cells are ofthe mature CD14−, CD83+ , HLA-DR^(hi) phenotype. FACSort® (BectonDickinson, San Jose, Calif.) is used to generate highly pure populationsof immature and mature DCs, based on their CD83− and CD83+ phenotypes,respectively. Macrophages are isolated from T cell-depleted fractions byplastic adherence for 1 h. After 24 h, cells are removed from the platesand placed in Teflon beakers for 3-9 d. T cells are further purifiedfrom the T cell-enriched fraction by removing contaminating monocytes,NK cells, and B cells.

EXAMPLE 28 Induction of Apoptotic Death and Phagocytosis of ApoptoticTumor Cells or SAg-Transfected Tumor Cells by DCs

Monocytes are infected with influenza virus in serum-free RPMI. Thesecells undergo viral-induced apoptotic death within 6-8 h. Cell death isconfirmed using the Early Apoptosis Detection kit (Kayima BiomedicalCo., Seattle, Wash.). As previously described, cells are stained withAnnexin V-FITC (Ann V) and propidium iodide (P1). Early apoptosis isdefmed by Ann V+/PI staining as determined by FACScan® (BectonDickinson). Five to eight h after infection, monocytes first externalizePS on the outer leaflet of their cell membrane, as detected with Ann V.By 8-10 h, these cells are TUNEL (Tdt-mediated dUTP-biotin nick-endlabeling) positive. It is not until 24-36 h that the majority of themonocyte population included trypan blue into the cytoplasm, anindicator of secondary necrosis. HeLa cells are triggered to undergoapoptosis using a 60 UV lamp (Derma Control Inc.), calibrated to provide2 mJ/cm²/s.

Induction and Detection of Apoptosis

Monocytes are infected with influenza virus in serum-free RPMI. Celldeath is assayed using the Early Apoptosis Detection kit (KayimaBiomedical). Briefly, cells are stained with Annexin V-FITC (Ann V) andpropidium iodide (P1). Early apoptosis is 14 defmed by AnnV+/PI-staining as determined by FACScan (Becton Dickinson). Cells fromthe 293 cell line are triggered to undergo apoptosis using a 60 UVB amp(Derma Control Inc.), calibrated to provide 2 mJcm⁻²s⁻¹.

Phagocytosis of Apoptotic Cells

Monocytes and HeLa cells are dyed red using PKH₂6-GL (Sigma Biosciences,St. Louis, Mo.), and induced to undergo apoptosis by influenza infectionand UV irradiation, respectively. After 6-8 h, allowing time for thecells to undergo apoptosis, they are cocultured with phagocytic cellsthat were dyed green using PKH67-GL (Sigma Biosciences), at a ratio of1:1. Macrophages are used 3-6 d after isolation from peripheral blood;immature DCs are used on days 6-7 of culture; and mature DCs are used ondays 10-11. Where direct comparison of cells is needed, cells areprepared from the same donor on different days. In blocking experiments,the immature DCs are preincubated in the presence of 50 μg/ml of variousmAbs for 30 mm before the establishment of cocultures. After 451 20 mm,FACScan® analysis is performed and double positive cells wereenumerated.

Coculture of DCs with Apoptotic Cells

Monocytes from HLA-A2.1-donors are infected with live orheat-inactivated influenza virus. Live influenza virus (Spafas Inc.) isadded at a final concentration of 250 HAU ml-1 (MOI of 0.5) for 1 h at37° C. Virus is heat-inactivated by treatment for 30 min at 56° C.before use. After washing, cells are added to 24-well plates in varyingdoses. After 1 h, contaminating non-adherent cells are removed and freshmedia is added. Following a 10 h incubation at 37° C., 3.3×10³uninfected DCs and 1×10⁶ T cells are added to the wells.

Antigen Pulsing of DC

Day 7 DC are incubated with freeze-thawed tumor lysates at a ratio ofthree tumor cell equivalent to one DC (ie., 3:1) in CM. After 18 hr ofincubation, DC are harvested, irradiated with rad (Gamma Cell 1000;Nordion, Kanata, Canada), washed twice in Hank's balanced salt solution(GIBCO), and in Hank's balanced salt solution.

EXAMPLE 29 Treatment of Tumor Bearing Animals with SAg-Transfected orSAg-Expressing DCs, Accessory Cells or S/D/t Cells: VaccinationProtocols and Treatment of Established Tumor

Immunotherapy

C57BL/6 mice are immunized once with irradiated, S/D/t cells (2×10⁶cells/mouse) 10-14 d post-immunization mice are challenged with 2×10⁷live tumor cell subcutaneously in the scapular region. Mice aremonitored on a regular basis for tumor growth and size. Mice with tumorsizes >3.5 cm were killed. All survivors were killed 40 dpost-challenge.

P10.9-B 16 Melanoma Model.

Mice are injected intra-footpad with 2×10⁵ F10.9 cells. Legs areamputated when the local tumor in the footpad is 7-8 mm in diameter.Post-amputation mortality is less than 5%. 2 d post-amputation mice areimmunized intraperitoneally with S/D/t cells followed by weeklyvaccinations twice, for a total of three vaccinations. Mice are killedbased on the metastatic death in the non-immunized or control groups(28-32 d post-amputation). Metastatic loads are assayed by weighing thelungs.

S/D/t cells: In Vivo Immunization and Tumor Challenge

B6 or BALB/c mice are immunized s.c. in the right flank with 1×10⁶MCA-207 or 1×10⁶ S/D/t cells, respectively, twice at 7-day intervals.Mice then are rechallenged 7 days after the last immunization with alethal dose of 1×10⁵ MCA-207 (for B6 mice) or 3×10⁵ MT-901 (for BALB/cmice) viable tumor cells by s.c. injections into the left flank. Thesize of the tumors is assessed in a blinded, coded fashion twice weeklyand recorded as tumor area (in square mm) by measuring the largestperpendicular diameters with calipers. Data are reported as the averagetumor area SEM (five or more mice per group).

Vaccination Protocol

B6 mice are s.c. immunized twice in a 2-wk interval with 10⁶ irradiated(15,000 rad) B16, B16 mixed with DCs (1/1: unfractionated cells fromovernight culture), or S/D/t cells or recombinant formalin fixedbacteria (10⁶ -10⁸). Ten days following the final immunization, eachgroup of mice is injected s.c. with varying doses (10⁴, 10 ⁵, or 10⁶cells/mouse) of viable B16. Tumor growth and survival time of each groupof mice are recorded. The size of the tumor in each mouse is measured intwo perpendicular dimensions with a Vernier caliper twice weekly aftertumor challenge. Tumor incidence is considered positive when the averagediameters of the tumor exceeded 3 mm.

In Vivo Immunization for Treatment of Pulmonary Metastases

B6 or BALB/c mice receive 1.5×10⁵ MCA-207 or 2×10⁵ MT-901 viable tumorcells, respectively, i.v. in the lateral tail vein to establishpulmonary metastases, as described. The mice then are immunized s.c.with, respectively, 1×10⁶ MCA-207 tumor lysate-pulsed S/D/t cells threetimes on days 3, 7, and 11 or 1×10⁶ MT-901 tumor lysate-pulsed S/D/tcells twice on days 3 and 7 after tumor injection and are killed on days14 and 17, respectively. Pulmonary metastases are enumerated on day 15(MCA-207) or 14 (MT-901). Data are reported as the mean number ofmetastases ±SEM (five or more mice per group).

In vitro Activation of LN T cells

B6 mice are immunized s.c. twice in a 2-wk interval on the flanks with2×10⁶ (10⁶/side) irradiated (15,000 rad) tumor, S/D/t cell preparation,or tumor mixed with DCs (1/1) suspended in 0.1 ml of HBSS. One weekafter the final immunization, inguinal LNs from each group of mice areharvested. LN cells from each group of mice are activated and expendedin culture using anti-CD3 plus IL-2. In brief, LN cells (3-4×10⁶cells/well) are activated on 24-well plates coated with anti-CD3 mAb(145-2C11) and incubated at 37° C. for 2 days. Alternatively, S/D/tcells (10⁴-10⁵/well) or exosomes (3-5 g) or recombinant bacteria(10⁶-10⁸/well) are incubated with the LN cells for 2 days and optionallywith low dose IL-2 for an additional 2 days. The activated cells aresuspended at 1-2×10⁵ cells/ml in CM containing IL-2 (4 U/ml) andincubated in gas-permeable culture bags (Baxter Healthcare, Deerfield,Ill.) for an additional 3 days. The derived LN T cells are harvested andused as effector cells for adoptive immunotherapy.

Adoptive Immunotherapy Models

For therapy of B 16 pulmonary metastases. B6 mice are injected i.v. with105 live B16 tumor cells in 1 ml of PBS to initiate pulmonarymetastases. Three days after tumor inoculation, mice are randomlydivided into several groups to receive treatments by i.v. injection of5×10⁷ cultured LN T cells suspended in 1 ml of PBS. On day 21 aftertumor inoculation, mice from each group are killed, and lungs areinsufflated with Fekete's solution. Lung metastases are counted. In someexperiments. tumor-bearing mice are i.p. administered IL-2 (15,000 U.twice/day for 5 days) following the adoptive transfer of cultured LN Tcells. For therapy of FBL-3 tumor. B6 mice are inoculated i.p. with5×10⁶ viable FBL-3 tumor cells on day 0. By day 5, the tumor isdisseminated, and mice are treated with cyclophosphamide (CY) at a doseof 180 mg/kg followed in 6 h by i.p. injection of cultured LN T cells(5×10 ⁷ cells/mouse) suspended in 0.5 ml of PBS. The tumor growth andthe survival time of each group of mice are monitored and recorded on aregular basis

Induction of anti-tumor activity by FC/MUC1.

Groups of 1 mice are immunized twice at 14-day intervals by subcutaneousinjection of 3×10⁵ DCs (0) or S/D/t cells represented by FC/MUC1cells.PBS is injected as a control (0). After 14 days, mice are challengedsubcutaneously with 2.5×10⁵ MC38/MUC1 cells. Tumors of 3 mm in diameterare scored as positive.

Immunization with FC/MUC1 for Prevention and Treatment of PulmonaryMetastases

Groups of 10 mice are injected twice with S/D/t cells represented byFC/MUC1cells or PBS and then challenged after 14 days with intravenousadministration of 1×10⁶ MC38/MUC1 cells. The mice are killed 28 daysafter challenge. Pulmonary metastases are enumerated after staining thelungs with India ink. Groups of 10 mice are injected intravenously with1×10⁶ MC38/MUC1 or MC38 cells. The mice are immunized with 1×10⁶ S/D/tcells representing FC/MUC1 cells or FC/MC38 at 4 and 18 days after tumorchallenge and then killed after an additional 10 days. Pulmonarymetastases are enumerated for each mouse.

Protection Assays

C57BL/6 mice are immunized with the indicated antigen-gene construct.Animals are challenged with tumors and evaluated for tumor survival asdescribed. Briefly, 7 days after the final immunization (day 0),immunized animals are challenged by intradermal injection in themid-flanks bilaterally with melanoma cells (2×10⁴) at two times the doselethal to 50% of the animals tested (LD50). Survival is recorded as thepercentage of surviving animals. Melanoma cells for injection are washedthree times in PBS. Injected cells were greater than 95% viable bytrypan blue exclusion. All experiments include five mice per group andwere repeated at least three times. Mice that became moribund werekilled according to animal care guidelines

EXAMPLE 30 DNA or RNA from SAg Transfected Tumor Cells, SAg TransfectedDCs and SAg Transfected DC/tc Hybrids for In Vivo Vaccination andTransfection of Naive DCs to Produce a DC Expressing SAgs and TumorAssociated Antigens

Plasmid DNA Vector

-   1. Genes from SAg Transfected Tumor Cells, SAg transfected DCs and    S/D/t cells are cloned by PCR to contain a partial or entire coding    region. In most cases, it is desirable to not include any sequence    5′ to the ATG or 3′ to the termination codon. PCR primers are    designed to contain a restriction site, such as BglII or BamHI.-   2. The PCR fragments are separated from unreacted oligomers and    template and then the fragment is cut with an excess of BglII for at    least 5 h. The DNA is Phenol extracted and ethanol-precipitated. The    purified cut fragment is resuspended in TE, pH 8.0 and ligated. to    BglII-digested V1J, which has been gel-purified and dephosphorylated    with calf intestinal alkaline phosphatase (CLAP), phenol-extracted,    ethanol-precipitated, and resuspended in TB, pH 8.0. A 6:1 molar    ratio of insert:vector in the ligation reaction is used.-   3. Competent E. coli cells (e.g., DH5, DH5a) are transformed with    the ligation reaction, plated on L-ampicillin plates and grown    overnight at 37° C. Colonies are screened by hybridization of plate    lifts to kinase-labeled PCR primer. Several hybridization-positive    colonies are selected and grown in overnight cultures for miniprep    purification.-   4. Miniprep DNAs, are prepared and cut with the appropriate    restriction enzymes to determine correct orientation of the gene in    the vector. At least three DNAs with the gene in the correct    orientation are selected to confirm by sequencing across the    ligation junctions. Sequencing primers are designed from the vector    sequence. Each primer is 30-50 bp from the restriction site (BglII    in the example), so that 10-20 bases within the vector can be read    as well as 150-200 bases within the gene. This amount of sequence    verifies orientation and give a reasonable estimate of the quality    of the PCR-generated gene.-   5. DNA preparations that have been sequence-verified 1/1000 in TB,    pH 8.0, are diluted and use to retransform competent E. Coli. Three    isolated colonies from the transformation plates are grown overnight    at 37° C., and used to make a −70° C. cell stock by adding 0.8 ml    fresh overnight growth to 0.2 ml sterile 80% (v/v) glycerol, mixing    well, and freezing on dry ice. The −70° C. stocks are used to    isolate plasmid DNA from remaining cells by miniprep procedures.    Miniprep DNA is cut again with the appropriate restriction enzymes,    and visualized on a gel to verify the construct. All subsequent    growth of cells for plasmid production are made from the −70° C.    frozen stock.

All constructs are tested in vitro to validate their ability to expressthe desired gene product. Plasmids purified by column (Wizard preps,Promega, Madison, Wis.) or by cesium chloride banding are used totransfect tissue-culture cells transiently. Protein expression isdetected by immunoblot. This check not only verifies expression but canvalidate the size and immunoreactivity of the gene product.

Characterization of Plasmid DNA Vectors

All constructs are tested in vitro to validate their ability to expressthe desired gene product. Plasmids purified by column (Wizard preps,Promega, Madison. Wis.) or by cesium chloride banding are used totransfect tissue-culture cells transiently. Protein expression isdetected by immunoblot. This check not only verifies expression but canvalidate the size and immunoreactivity of the gene product.

Cell Growth and Transfection

-   1. DC /tumor cell hybrids, at 0.8-1.5×10⁶ cells/100 mm plate in    Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10%    heat-inactivated fetal bovine serum, 20 mM HEPES, 4 mM L-glutamine,    and 100 μg/ml each of penicillin and streptomycin, and incubate at    37° C. in 5% CO₂ for 18 h.-   2. The construct to be tested is cotransfected with 10 μg/plate and    10 g of V1J-CAT using a calcium phosphate procedure or other methods    given in Example 1.-   3. Five hours after transfection, the cells are shocked in 15% (v/v)    glycerol in PBS, pH 7.2, for 2.5 mm.-   4. Cultures are harvested 72 h after transfection by washing the    plates twice with 10 ml of cold PBS, pH 7.2, then adding 5 ml of    cold TEN buffer and scraping.-   5. Pellet cells and use immediately or store at −70° C. for    subsequent analysis.    Immunoblot Analysis-   1. Cell pellets are lysed in Single Detergent Lysis Buffer, and    sonicate on ice (2-15 s bursts) to reduce viscosity.-   2. Cell debris is removed by sedimentation and determine soluble    protein concentrations of the supernatants by the Bradford method.-   3. Equal loadings of soluble cell protein per lane are run on    SDS-polyacrylamide gel and transfer the proteins to Immobilon P    (Millipore, Bedford, Mass.) membrane.-   4. Western blots are incubated overnight with an appropriate    dilution of the antibody to the gene product being tested, followed    by a 1.5-h reaction with a 1:1000 dilution of peroxidase-conjugated    secondary antibody. Develop blots using the ECL kit (Amersham,    Arlington Heights, Ill.).    Large-Scale DNA Preparations-   1. Expression vectors are grown in E. coli strain DH5 with vigorous    aeration in 500 ml growth medium/1-L shake flask. V1J constructs are    grown overnight to saturation.-   2. Cells are harvested and lysed by a modification of the alkaline    SDS procedure. The modification consists of increasing the volumes    threefold for cell lysis and DNA extraction.-   3. DNA is purified by double banding on CsC1/ethidium bromide    gradients.-   4. The ethidium bromide is removed by 1-butanol extraction, and the    resulting DNA is extracted with phenol/chloroform and precipitated    with ethanol.-   5. DNA in TE for transfections is resuspended and in 0.9% NaCl for    injection into mice.-   6. The concentration and purity of each DNA preparation is    determined by A 260/280 readings. The 260/280 ratios are >1.8.-   7. DNA is stored in small aliquots at −20° C.

EXAMPLE 31 DNA Immunization in vivo

-   1. Animals are housed in an American Association for the    Accreditation of Laboratory Animal Care (AAALAC) accredited facility    or other national facility and cared for in accordance with the    Guide for the Care and Use of Laboratory Animals. Prior to bleeding,    or administration of anesthetic or inoculation, animals are in good    physical condition and free from stress.-   2. For administration of DNA vaccines, animals are anesthetized by    ip injection of a solution containing ketamine and xylazine (50 and    20 μg/g body wt, respectively) in a total volume of 0.3 ml of    saline. Alternatively, transiently immobilize mice for a sufficient    period of time to administer an im injection by allowing inhalation    of metophane. Larger animals, such as ferrets or nonhuman primates,    are anesthetized using ketamine (30 mg/kg)/xylazine. (2    mg/kg)/atropine (1 mg/kg) or ketamine (10 mg/kg), respectively.-   3. Fully anesthetized animals are prepared for injection by flooding    and swabbing the injection site with ethanol (70%). This provides    sterilization and, for small animals, such as mice, facilitates    visualization of the muscle groups. To visualize small muscles    further, fur around the injection site is shaved followed by ethanol    swabbing, or a short incision can be made to permit direct    observation of the muscle. In the latter case, the incision is    sutured after inoculation.-   4. DNA vaccines are administered in saline solution alone or    together with a facilitator that induces muscle generation or    regeneration. Facilitators are used in animals that may not    necessarily be used in humans. For mice, volumes of up to about 50    μL are injected into each quadriceps muscle using a disposable    insulin syringe equipped with a 27-gauge needle and having a    capacity of 0.3 ml.-   5. DNA vaccines are also administered using particle bombardment    technology. Plasmid DNA is coated onto gold beads and propelled    directly into tissue. Genetic immunization is accomplished by    biolistic bombardment using methods similar to those recently    described. Briefly, DNA-coated gold particles are prepared by    combining 50 mg of 0.95 um gold beads and 100 1 of 0.1 M-   spermidine and sonicating for 5 s. Plasmid DNA (100 μg) and CaCl    (200 μl) are added sequentially to the beads spinning in a vortex;    mixer. This mixture is allowed to precipitate at room temperature    for 5-10 mm. The bead preparation is then centrifuged (10,000 r.p.m.    for 30 s) and washed 3 times in cold ethanol before resuspension in    7 ml of ethanol to give a final concentration of 7 mg gold per    milliliter. The solution is then loaded into Tefzel tubing    (Agracetus, Middleton, Wis.) and allowed to settle for 5 mm. The    ethanol is removed and the beads are attached to the side of the    tubing by rotation at 20 r.p.m. for 30 s and N₂ dried. The dried    tubing lined with beads is then cut into 0.5-inch sections and    stored for use with desiccant in parafilm-sealed vials. Animals are    vaccinated by delivery of two shots (each shot consisted of 0.5 m4j    gold beads in 0.5 inch of tubing) to the shaved abdominal region    using the Accell gene delivery device (Agracetus) at a discharge    pressure of 400 p.s.i. This delivers approximately 1.00μg/DNA per    shot. Animals are immunized with various plasmids In some    experiments, particles are coated with the pGREEN LANTERN-1 plasmid    (Gibco BRL, Gaithersburg, Md.), which contains the “humanized”    reporter gene encoding GFP from the Aequorecia Victoria jellyfish.    This gene encodes a naturally fluorescent protein requiring no    substrates for visualization.    Formulation of DNA Vaccine:

Saline is the preferred solvent. However, plasmid DNA may also beadministered in various other buffer formulations and cationic lipidformulations. Facilitators include anesthetics, such as bupivacaine, andtoxins, which are used in conjunction with DNA vaccines. Conventionaldelivery vehicles are used which facilitate internalization of DNA bycells, protect DNA from digestion by extracellular nucleases, or effecta slow release of DNA; adjuvants are coadministered to provide anadditional stimulus for the immune system.

Dosage and Injection Regimen:

DNA vaccines are effective across a broad dosage range. Protectiveefficacy is achieved with submicrogram amounts of DNA. With respect tohumoral immune responses against HA, there is a direct correlationbetween magnitude of antibody responses and dose of DNA between 10 ngand at least 100 g. However, perhaps owing to viscosity of the solutionand/or distribution of the inoculum in the muscle, administration of DNAat concentrations in excess of 2-4 mg/ml results in decreasedimmunogenicity with some antigens. Therefore, in mice, doses in excessof 200 g are not practical by im injection. The number of injectionsalso directly correlates with magnitude of immune responses (up to atleast three). For the influenza model in mice, we have found that threeinjections given at 3-wk intervals yield optimal protection. It islikely, however, that dosing and regimen will need to be optimized foreach gene and challenge model.

Site of injection:

Injection of plasmid DNA into muscle cells is far superior to other celltypes in their capacity to internalize DNA and/or express reporterproteins in vivo. However, immune responses also have been generatedafter id and iv routes of DNA injection. In addition, particlebombardment of DNA results in the transfection of dermal and epidermalcells leading to the generation of immune responses. The relativeeffectiveness of these different routes of delivery has yet to be testedrigorously. However, direct im injection generates a protective immuneresponses at doses (100 ng to 1 g) and is preferred in the range used byparticle bombardment.

EXAMPLE 32 Pulsing DCs with RNA from SAg Producing Bacteria or S/D/tCells

Total RNA is isolated from SAg producing bacteria or S/D/t cells bystandard methods. Pulsing DCs with RNA from SAg producing bacteria,S/D/t cells or SAg transfected tumor cells is performed in serum-freeOpti-MEM medium (GIBCO BRL) for tumor extracts with the followingmodification RNA (25 g in 250 1 Opti-MEM medium) and DOTAP (50 g in 2501 Opti-MEM medium) are mixed in 12×75 mm polystyrene tubes at roomtemperature for 20 mm. The complex is added to the DCs (25×10⁶ cells/ml)and incubated 37° C. in a water bath with occasional agitation for 25mm. The cells are washed twice and resuspended in PBS (10⁵ RNA pulsedDCs in 500 l PBS/mouse) for intraperitoneal immunizations. PBS, B16extract from 10⁵ cells in PBS, or DCs prepared as described above areinjected intraperitoneally in a volume of 500 l.

EXAMPLE 33 PolyA-Cellular RNA from S/D/t cells or DCs Transfected withSAg: Preparation and Immunization Protocols

Total RNA is isolated from actively S/D/t cells given above as follows.Briefly, 10⁷ cells are lysed in 1 ml of guanidinium isothiocyanate (CT)buffer (4 M guanidinium isothiocyanate, 25 mM sodium citrate, pH 7.0;0.5% sarcosyl, 20 mM EDTA, 0.1M 2-mercaptoethanol). Samples are vortexedfollowed by sequential addition of 100 l 3M sodium acetate, 1 ml watersaturated phenol and 200 l chloroform/isoamyl alcohol (49:1).Suspensions are vortexed and placed on ice for 15 mm. The tubes arecentrifuged at 10,000 g, 4° C. for 20 min and the supernatant iscarefully transferred to a fresh tube. An equal volume of isopropanol isadded and the samples are placed at −20° C. for at least 1 h. RNA ispelleted by centrifugation as above. The pellet is resuspended in 300 lGT buffer which is then transferred to a microcentrifuge tube. RNA isre-precipitated by adding an equal volume of isopropanol and placing thetube at −20° C. for at least 1 h. Tubes are microcentrifuged at highspeed at 4° C. for 20 mm. Supernatants are decanted and pellets arewashed once with 70% ethanol. Pellets are allowed to dry at RT and thenresuspended in TB (10 mM Tris-HCl, 1 mM EDTA, pH 7.4). Possiblecontaminating DNA is removed by incubating RNA in 10 mM MgCl₂, 1 mM DTTand 50 U/ml RNase free DNase (Boehringer-Mannheim, Indianapolis, Ind.)for 15 min at 37° C. The solution is adjusted to 10 mM Tris, 10 mM EDTA,0.5% SDS and 1 mg/ml Pronase (Boehringer-Mannheim) followed byincubation at 37° C. for 30 mm. Samples are extracted once withphenol-chloroform and once with chloroform, and RNA was thenre-precipitated in isopropanol at −20° C. After centrifugation thepellets are washed with 70% ethanol, air dried, and resuspended insterile water. Total RNA is quantitated by measuring OD at 260 and 280nm. OD 260/280 ratios are typically 1.65-2.0. RNA is stored at −70° C.PolyA+ RNA is either isolated from total RNA using Oligotex (Qiagen,Chatsworth, Calif.) or directly from tissue culture cells using theMessenger RNA Isolation kit (Stratagene, La Jolla, Calif.) as permanufacturer's protocols.

Production of In vitro Transcribed RNA

The 1.9-kb EcoR1 fragment containing the coding region and 3′un-translated region is cloned into the EcoR1 site of pGEM4Z (Promega,Madison, Wis.). Clones containing the insert in both the sense andanti-sense orientations are isolated and large scale plasmidpreparations are made using Maxi Prep Kits (Qiagen). Plasmids arelinearized with BamHl for use as templates for in vitro transcription.Transcription is carried out at 37° C. for 34 h using the 5P6 MEGAscriptIn vitro Transcription Kit (Ambion, Austin, Tex.) per manufacturer'sprotocol and adjusting the GTP concentration to 1.5 mM and including 6mM m7G(5′)ppp(5′)G cap analogue (Ambion). Template DNA is digested withRNase free DNase I and RNA is recovered by phenol/chloroform andchloroform extraction followed by isopropanol precipitation. RNA ispelleted by microcentrifugation and the pellet is washed once with 70%ethanol. The pellet is air-dried and resuspended in sterile water. RNAis incubated for 30 mm at 30° C. in 20 mM Tris-HCl, pH 7.0, 50 mM KCl,0.7 mM MnCl₂, 0.2 mM EDTA, 100 μg/ml acetylated BSA, 10% glycerol, 1 mMATP and 5,000 U/ml yeast poly(A) polymerase (United States Biochemical,Cleveland, Ohio). The capped, polyadenylated RNA is recovered byphenol/chloroform and chloroform extraction followed by isopropanolprecipitation. RNA is pelleted by microcentrifugation and the pellet iswashed once with 70% ethanol. The pellet is air-dried and resuspended insterile water. RNA is quantitated by measuring OD at 260 and 280 nm andstored at −70° C.

Pulsing of Antigen-Presenting Cells, Accessory Cells DCs. Tumor Cells orDC/Tumor Cell Hybrids with RNA Derived from S/D/t cells

Pulsing of cells with RNA is routinely performed in serum-free Opti-MEMmedium (GIBCO BRL). Cells are washed twice in Opti-MEM medium. Cells areresuspended in Opti-MEM medium at 25×10⁶ cells/nil and added to 15 mlpolypropylene tubes (Falcon). The cationic lipid, DOTAP, (BoehringerMannheim) is used to deliver RNA into cells. RNA (in 250-500 l Opti-MEMmedium) and DOTAP (in 250-500 p. 1 Opti-MEM medium) are mixed in12×75-mm polystyrene tubes at room temperature (RT) for 20 mm. Theamount of polyA+ RNA or IVT RNA used is 5 g and the amount of total RNAused is 25 g. The RNA to DOTAP ratio is 1:2. The complex is added to theAPC (2-5×10⁶ cells) in a total volume of 2 ml and incubated at 37° C. ina water-bath with occasional agitation for 2-4 h.

EXAMPLE 34 In vivo Immunization with RNA derived from “S/D/t cells” orSAg-Transfected Tumor Cells

Preparation of mRNA for Transfection

DNA is linearized downstream of the poly A tail with a 5-fold excess ofPstI. The linearized DNA is then purified with two phenol/chloroformextractions, followed by two chloroform extractions. DNA is thenprecipitated with NaOAc (0.3M) and 2 volumes of EtOH. The pellet isresuspended at about 1 mg/ml in DEP-treated deionized water.

A transcription buffer is prepared, comprising 400 mM Tris. HCl (pH8.0), 80 mM MgCl₂, 50 mM DTT, and 40 mM spermidine. The followingmaterials are added in order to one volume of DEP-treated water at roomtemperature: 1 volume T7 transcription buffer; rATP, RCTP, and rUTP to 1mM concentration; rGTP to 0.5 mM concentration; 7 g(5′)ppp(5′)G capanalog (New England Biolabs, Beverly, Mass.) to 0.5 mM concentration;the linearized DNA template to 0.5 mg/ml concentration; RNAsin (Promega,Madison, Wis.) to 2000 U/ml concentration; and T7 RNA polymerase (N.E.Biolabs) to 4000 U/ml concentration.

This mixture is incubated for 1 hour at 37° C. The successfultranscription reaction is indicated by increasing cloudiness of thereaction mixture.

Following generation of the mRNA, 2U RQ1 DNAse (Promega) per microgramof DNA template used is added and was permitted to digest the templatefor 15 minutes. Then, the RNA is extracted twice with chloroform/phenoland twice with chloroform. The supernatant is precipitated with 0.3MNaOAc in 2 volumes of EtOH, and the pellet is resuspended in 100 mu 1DEP-treated deionized water per 500 l transcription product. Thissolution is passed over an RNAse-free Sephadex G50 column (BoehringerMannheim #100 411). The resultant mRNA is sufficiently pure to be usedin transfection of vertebrates in vivo.

mRNA Vaccination in vivo

A liposomal formulation containing mRNA coding for the SAg/tumorassociated antigen protein prepared and is inserted into the plasmidpXBG in A volume of 200 l of a formulation is prepared containing 200μg/ml of S/D/t cell-derived mRNA and 500 μg/ml 1:1 DOTAP/PE in 10%sucrose is injected into the tail vein of mice 3 times in one day. Atabout 12 to 14 h after the last injection, a segment of muscle isremoved from the injection site, and prepared as a cell lysate accordingto Example 7. The S/D/t cell-derived specific protein is identified inthe lysate.

Severe combined immunodeficient (SCID) mice (Molecular BiologyInstitute, (MBI), La Jolla, Calif.) were reconstituted with adult humanperipheral blood lymphocytes by injection into the peritoneal cavityaccording to the method of Mosier (Mosier et al., Nature 335:256(1988)). The mice were maintained in a P3 level animal containmentfacility in sealed glove boxes. mRNA coding for the S/D/t cell-derivedproteins is prepared by obtaining the S/D/t cell gene in the form of aplasmid removing the gene from the plasmid; inserting the gene into thepXBG plasmid for transcription; and purifying the transcription productS/D/t cell-derived mRNA. The S/D/t cells mRNA is then incorporated intoa formulation and 200 l tail vein injections of a 10% sucrose solutioncontaining 200 μg/ml S/D/t cell RNA and 500 μg/ml 1:1 DOTAP:DOPE (inRNA/liposome complex form) were performed daily on experimental animals,while control animals were likewise injected with RNA/liposome complexescontaining 200 μg/ml yeast tRNA and 500 μg/ml 1:1 DOTAP/DOPE liposomes.At 2, 4 and 8 weeks post injection, biopsy specimens are obtained frominjected lymphoid organs and prepared for immunohistochemistry.

A volume of 200 l of the formulation, containing 200 μg/ml MRNA fromS/D/t cells, and 500 μg/ml 1:1 DOTAP:DOPE in 10% sucrose is injectedinto the tail vein of the human stem cell-containing SCID mice 3 timesin one day. Following immunization, the mice are challenged by tumorinoculation.

The full-length sequence for the cDNA of the S/D/t-derived gene isobtained and ligated to BgIII linkers and then digested with BgIII. Themodified fragment is inserted into the BgIII site of pXBG. S/D/t-derivedprotein is transcribed and purified mRNA is incorporated into aformulation. Balb 3T3 mice are injected directly in the tail vein with200 l of this formulation, containing 200 μg/ml of S/D/t-derived mRNA,and 500 μg/ml DOTAP in 10% sucrose.

EXAMPLE 35 Preparation of “String of Beads” Tumor Antigens forTransfection of SAg-Transfected DCs, Other Accessory Cells, or TumorCells

Generation of rAd

All cell lines were maintained in Iscove's modified Dulbecco's medium(IMDM) (Scromed, Berlin) supplemented with 4% fetal calf serum(FlyClone), penicillin (110 international units/ml; Brocades Pharma,Leiderdorp, The Netherlands). and 2-mercaptoethanol (20 μM) at 37° C. ina 5% CO₂ atmosphere. The adenoviral vector construction adapter plasmidpMad5 is derived from plasmid pMLPI0 as follows. pMLPI0-lin isconstructed by insertion of a synthetic DNA fragment with unique sitesfor the restriction endonucleases MluI, SplI, SnaBI, SpeI, Asull, andMunI into the Hindlll site of pMLP10. Subsequently, the adenovirus BglIIfragment spanning nucleotides 3328 8914 of the AdS genome is insertedinto the Munl site of pMLP-lin. Finally, the Sall-BamH1 fragment isdeleted to inactivate the tetracycline resistance gene, resulting inplasmid pMad5. A mini-gene cassette vector, pMad5-0. is generated byligation of the annealed and phosphorylated double-strandedoligonucleotides 1a/b and 2a/b into the MluI and SpeI sites of pMad5.This cloning step leads to elimination of the original MluI and SpeIsites and to creation of a small ORF, which essentially consists of astart codon, the sequence SEOKLISEEDLNN, a human c-Myc-derived sequence,which is recognized by mAb 9E10 and a stop codon. A small “stuffer”sequence. flanked by newly generated MluI and SpeI sites, is presentbetween the start codon and the c-Myc sequence.

pMad5-1 and -2, each of which harbor a multi-epitope encoding minigene.are constructed by unidirectional cloning of the followingdouble-stranded **oligonucleotides into pMad5-0, which had been cleavedwith MluI and SpeI. pMad5-I. After each cloning step, the sequence ofthe inserts is verified by DNA sequencing. Expression of these minigenesis driven by the Ad5 major late promoter, which in this configuration islinked to the AdS immediate early enhancer, resulting in immediate earlyexpression of the minigenes. rAds are generated through in vivohomologous recombination in the Ad5E1-transformed helper cell line 911between plasmid pJMI7. containing the sequence of the AdS mutant d1309,and either of the plasmids pMad5-1 or pMad5-2. 911 cells are transfectedwith 10 g of plasmid pJMI7 in combination with 10 g of either pMad5-1 orpMad5-2. The rAds are plaque-purified three times, after which theclonal rAds are propagated in 911 cells, purified by double cesiumchloride density gradient centrifugation. and extensively dialyzed. Thepresence of replication-competent adenoviruses is routinely checked byinfection of Hep-G2 cells. The viral stocks were stored in aliquots with10% glycerol at −80° C. and titered by plaque assay using 911 cells.

Further Transfection of SAg-Transfected DCs, Accessory Cells, or TumorCells

In short, 100 ng of plasmid DNA encoding Ad5LI, HPV 16 E7, murine p53 orthe influenza-matrix protein are transfected into 1×10⁴ SAg-transfectedDCs, accessory cells or tumor cells. The transfected cells are incubatedin 100 μl of IMDM containing 8% fetal calf serum for 48 h at 37° C.,after which 1500-500 CTL ??? in 25 μl of IMDM containing 50 Cetus units(=300 international units) of recombinant interleukin-2 (Cetus) areadded. After 24 h, the supernatant is collected, and its tumor necrosisfactor (TNF) content is determined by measuring its cytotoxic effect onWEHI-164 clone 13 cells.

EXAMPLE 36 Production of Exosomes from DCs Expressing SAg and TumorAssociated Antigens and Normal Hepatocytes

Exosome Isolation

SAgs or tumor associated antigens are transfected into tumor cells, DCs,or DC/tc hybrids by methods disclosed herein.. The SAg-encoding nucleicacid is provided with sorting sequences which route the translatedprotein to the endoplasmic reticulum and thereupon to secretory vesiclesor exosomes. Alternatively, tumor cells, DCs or DC/tc are incubated18-20 hours with tumor peptides or SAgs. DCs supernatants are harvested,centrifuged (at 4° C.) at 300 g for 20 mm and then at 10,000 g for 30min (to eliminate cell debris). Exosomes are then pelleted at 100,000 gfor one hour, and washed once in a large volume of PBS (over 100-foldthe final volume of resuspension of the exosomes). The proteinconcentrations in exosome preparations is measured by Bradford assay(BioRad). The slightly acidic pH transiently induced by the acid peptideelution increases the amounts of exosomes produced by DCs. Three to fiveg of exosomes are routinely obtained from 5-10×10⁵ DCs in 18-20 hours.Exosomes containing LDL, oxyLDL, apolipoproteins, LDL receptors andoxyLDL receptors are obtained from normal hepatocytes by a methodsimilar to that described above for dendritic cells and sicklederythrocytes as in Example 6.

Mice and Tumor Cell Lines for Exosome Trials

DBA/2J (H-2^(d)) and BALB/c (H-2^(d)) female mice 6-8 weeks of age areraised in pathogen-free conditions. P815 (H-2^(d)) is amethylcholanthrene induced mastocytoma, syngeneic with DBA/2. TS/A(H-2^(d)) is a spontaneously-arising undifferentiated mammaryadenocarcinoma, syngeneic with BALB/c. All tumor cell lines aremaintained in RPMI 1 640 supplemented with 10% endotoxin-free fetal calfserum (Gibco BRL), 2 mM L-Glutamine, 100 U/ml penicillin, 100 μg/mlstreptomycin, essential amino acids and pyruvate.

Experimental Mouse Models for Exosome Trials

Twice the minimal tumorigenic dose of tumor cells (5×10⁵ P815, 10⁵ TS/A)is inoculated intradermally in the upper right flank of DBA/2 and BALB/cmice, respectively. Animals with established tumors at days 3-4 forTS/A, or days 8-10 for P815, are immunized with a single intradermalinjection of 3-5 g of exosomes per mouse in the lower ipsilateral flank.The tumor size is monitored biweekly and mice are sacrificed whenbearing ulcerated or huge tumor burdens. All experiments are performedtwo to three times using individual treatment groups of five mice pergroup.

EXAMPLE 37 Bacterial Constructs for the Expression of SAgs Linked toGalactosylceramides, α-Gal Epitope, Peptidoglycans, Lipopolysaccharidesand □1,3-Glucans

Nucleic acids encoding SAgs may be transfected into bacteria whichnaturally synthesize and express fundamental recognition units forinnate immunity. Some of these moieties such as monogalactosylceramidesand α-galactosylceramides are potent immunogens and induce anti-tumoractivity. The addition of the SAg and a dominant tumor associatedepitope coexpressed with these natural bacterial constructs andadministered to a tumor bearing host would promote a potent tumorspecific response. The system described uses S. carnosus as a modelbacterial system to express a SAg peptide and dominant tumor epitope.

Expression Vectors for Surface Display.

The shuttle vector constructed pSPPmABPXM consists of the followingparts: (i) the origin of replication for E. coli and the 13-lactamasegene giving ampicillin resistance for transformed E. coli cells; (ii)the origin of replication for phage f1; (iii) the origin of replicationfrom S. aureus and the chloramphenicol acetyltransferase gene forstaphylococcus expression; (iv) the promoter, signal sequence, andpropeptide sequences from the S. hyicus lipase gene construct, optimizedfor expression in S. carnosus; (v) a multicloning site containing threeunique recognition sites for restriction endonucleases; (vi) a genefragment encoding a serum ABP from streptococcal protein G; and (vii)gene fragments encoding the cell wall-anchoring regions X and M fromstaphylococcal protein A.

As a model system, the surface display of SAg staphylococcal enterotoxinB. substitutes for place the 80 amino acid malaria peptide M3 fromfalciparum blood stage antigen Pfl55/RESA.. A plasmid vector,pSPPM3ABPXM is constructed, in which a gene fragment encoding SEBinstead of M3 is introduced between the propeptide region and the ABPsequence of plasmid pSPPmABPXM. An oligonucleotide linker(5′AGCTTGGCTGTTCCGCCATGGCTCGAG-3′ with complementary sequence) isinserted into the HindIII site of plasmid pSZZmpISX thus creatingadditional NcoI and XhoI recognition sites downstream of the HindIIIsite in the resulting vector, pSZZmpI8XhoXM. A gene fragment encoding a198-amino-acid ABP from the serum albumin binding region ofstreptococcal protein G is generated by a PCR (primers respectively)5′-CCGAATTCAAGCTTAGATGCTCTAGCAAAAGCCAAG-3′ and5′-CCCCTGCAGTTAGGATCCCTCGAGAGGTAAAATTTCATC-3′

with plasmid pSPGI as template sequenced in plasmid pRIT28 bysolid-phase DNA sequencing and HindIII-XhoI subcloned in framedownstream of the mp18 multilinker of pSZZmp18XhoXM. yielding plasmidpSZZmpI8ABPXM. An M3-encoding gene fragment was BamHI-HindIII subclonedfrom plasmid pRIT28EM3DAStop into pSZZmpI8ABPXM, yielding plasmidpSZZM3ABPXM. Plasmid pLipPS17 is constructed from pLipPSlk theintroduction of a BsmI recognition site in the beginning of the lipasesignal sequence. a Bc/I site at the end of the signal sequence and aBglII site at the end of the propeptide-encoding region by site-directedin vitro mutagenesis. A gene fragment constituting almost the entire S.carnosus vector pLipPSI except for a fragment encoding the C terminus ofthe propeptide and the majority of the mature lipase from S. hyicus isisolated by SalI-Hind III digestion and ligated to the E. Co/i plasmidpRIT28. which had previously been cut with the same restrictionendonucleases. The resulting plasmid. designated pSDLip. contained theorigin of replication for both E. coli and S. aureus. To restore theC-terminal region of the lipase propeptide, a gene fragment encoding theC-terminal part is generated by PCR amplification with theoligonucleotides 5-CCGAATTCTCGAGGCTCCTAAAGAAAATAC-3′ and5′-CCAAGCTTGGATCCTGCGCAGATCTTGGTGTTGGTTTTTTG-3′as upstream and downstream primers. respectively, with plasmid pLipPS17as template. This amplification introduced upstream EcoRI and XhoI sitesand downstream FspI. BamHI. and HindIII recognition sequences bynoncomplementary sequences in the PCR primers. The gene fragmentencoding the C-terminal propeptide region was EcoRI-HindIII suncloned topRIT28 to verify a correct sequence by solid-phase DNA sequencing andthereafter XhoI-BamHI transferred to SalI-BaniHI-restricted pSDLip. Theresulting plasmid. pSPP is HindIII restricted, filled in with Klenowpolymerase. and religated to yield plasmid pSPPDHind. which encodes thesignal peptide and the complete propeptide of the S. hyicus lipase withtranscription from a promoter region suitable for overproduction in S.carnosus.

EXAMPLE 38 Gene Transfer for Expression of an Mono orDigalactosylceramide by Transfection with a Cosmid Genomic LibraryPrepared from a Cell Line in which the Specific Glycosylceramide isHighly Expressed

The deliberate transfer of mono or digalactosylceramide expression intumor cells is achieved by transfection with a cosmid DNA libraryprepared from Fabry's cells in which the mono or digalactosylceramide ishighly expressed. This model demonstrates a general method fortransferring glycosyltransferase genes and other factors necessary forthe expression of glycosphingolipid antigens. The recipient tumor cellscontain mono or digalactosylceramide and the direct precursor,lactosylceramide. The transfected cells express mono ordigalactosylceramide detected both chemically and immunologically andcontained human DNA detected by an Alti sequence probe.

Cells and antibodies: Fabry's cells or normal cells with anα-galactosidase deficiency and tumor cells including but not limited toneuroblastoma cells are used. Anti-galactosyl ceramide monoclonalantibody is prepared. Total DNA is prepared from Fabry's cells isexcised by MboI and ligated by Bam HI-treated cosmid vector PCV 108,which has the SV40 promotor fused to the neomycin phosphotransferasegene. The target DNA for cosmid cloning is purified by gelelectrophoresis between 30-40 KB size. In vitro packaging is made withan extract of lysogenic bacteria and propagated in E. coli. as describedelsewhere. Transfection and Selection of Galactosylceramide Expression:Cosmid library DNAs are transfected into various cells using the calciumphosphate DNA precipitation technique with the addition of a glycerolshock after a 6 hour incubation. Galactosylceramide selection is started2 days later at 400 μg/ml concentration. The expression ofgalactosylceramide in the original Fabry's cell and the transfectedtumor cells was determined by cytofluorometry (FACS II), in whichFITC-conjugated anti- mono or digalactosylceramide antibody is used.Glycolipids in transfected cells are analyzed after cells were extractedin chloroform-methanol (2:1 and 1:1 v/v). The neutral glycolipidfraction is prepared by an acetylation procedure. The glycolipid profileis confirmed on HPTLC, followed by immunostaing with anti- mono ordigalactosylceramide antibody.

EXAMPLE 39 Staphylococcal Collagen Binding Adhesin Nucleic AcidsTransfected into SAg Transfected Tumor Cells, SAg Transfected DCs orAccessory Cells and S/D/t Cells

Collagen gene fragments from S. aureus strain FDA 574 are overexpressedin E. coli using the vector pQE-30 (QIAGEN inc. Chatworth, Calif.).Recombinant proteins expressed from this vector contain an NH₂-terminaltail of six histidine residues. The gene named cna encoding a S.aureuscollagen adhesin is isolated from a S. aureus genomic library cloned andsequenced. The cna gene encodes a 1185 amino acid polypeptide. Thededuced amino acid sequence reveals several structural characteristicssimilar to previously described Gram-positive bacterial cell surfaceproteins.

Plasmids expressing cna gene fragments are produced as follows.Recombinant S. aureus collagen adhesin fragments are overexpressed in E.coli using three different prokaryotic expression systems. The aminoterminus including the entire A domain is amplified from S. aureus FDA574 chromosomal DNA using PCR together with primers CNA 20 and CNA 21.The amplified 1.6-kb cna gene fragment is cleaved with EcoRI and PstI,gel purified and ligated to the prokaryotic expression vector pKK223-3obtained from Pharmacia LKB Biotechnology to create plasmid pKK1.5.Expression vector pKK223-3 contains an IPTG-inducible tac promoteradjacent to a consnesus Shine-Dalgarno ribosomal binding site. However,this vector lacks an initiation codon; therefore, the DNA to beexpressed must contain an appropriate start codon. In order to expressan internal cna fragment, a DNA linker sequence containing an ATG startcodon is synthesized. Two partially complementary ologonucletides, JPI(5′AATTACCATGGAATTCCTGCA-3′) and JP2 (5′-TGGTACCTTAAGG-3′), are heatedto 70° C. and slowly cooled to allow annealing. Once annealed, thedouble-stranded linker is phosphorylated by the addition of ATP and T4polynucleotide kinase. The DNA linker contained EcoRI and PstIrestriction sites at the 5′- and 3′-termini, respectively. These sitesare used to insert the linker onto pKK223-3. A 2.9-kb EcoRI/PstI DNAfragment, originally isolated from lambdaGT11 clone pCOL11 was ligatedto vector pKK223-3 to create plasmid pKK2.9. The collagen adhesinfragment encoded by pKK2.9 contains three repeated domains (B1, B2, andB3), the carboxyl terminus and downstream sequences. The plamidcontaining the collagen adhesin is transfected into DTES by methods inExample 1 and 3 and expression of the transduced gene is monitored byImmunoblots (Example 33).

EXAMPLE 40 Transfection of Nucleic Acids Encoding SAgs in Combinationwith Nucleic Acids the Promote Apoptosis Induction or Predispose toApoptosis

SAgs expressed in apoptotic tumor cells or tumor cell/DC hybrids areingested by DCs which present them to the immune system in more whichevokes a potent immune response to the tumor associated antigens. Theapoptotic cell is also one which is overexpresses a GalCer such as onewith a natural or acquired α-galactosidase deficiency or from a patientwith Fabry's Disease. The apoptotic stimulus can be produced byconcordant influenzal infection, radiation or chemotherapy. In addition,it may be inducible by an exogenous source such as TNF if the cell ispredisposed by transfection of an potent inhibitor of NF-κB such as amodified form of IκBa. Additional stimuli to apoptosis are provided bynumerous well established activators (caspase 9) or initiators (caspase8) of the caspase system or the CD95 TNFR network. Having undergoneapoptosis, the SAg transfected, GalCer overproducing cell is nowingested by DCs which are cross-primed to present the tumor antigens andthe GalCer in the context of SAg stimulation resulting in a potentantitumor response. Methods and protocols for SAg transfection are givenin Example 1 and for priming of DCs in Example 27-28 The apoptotictransfectants are used as a preventative or therapeutic antitumorvaccine by protocols in Example 15, 16, 18-23 and 29. They are alsouseful ex vivo to a population of tumor specific effector T cell or NKTcells for use in the adoptive immunotherapy of cancer (Examples 2-5, 7,15, 16, 18-23, 29).

EXAMPLE 41 Preparation and Isolation of Glycosphingolipids andVerotoxins Galabiosylceramide, Globotrioslceramides andGlobotetraosylceramide

Globotrioslceramides (GB3) and globotetraosylceramide (Gb4) are purifiedfrom human renal tissue. Briefly, the chloroform/methanol tissue extractis first applied on a Bio-Sil A (Bio-Rad) silica column in chloroform.The column is extensively washed with chloroform, and neutralglycolipids are eluted with acetone/methanol, 9:1 (vol/vol). The neutralglycolipid fraction is then applied on a second Bio-Sil A column inchloroform/methanol, 98:2 (vol/vol). Glycolipids are then resolved witha linear solvent gradient comprising equal weights ofchloroform/methanol 15:1 (vol/vol), to chloroform/methanol, 4:1(vol/vol). Galabiosylceramide (Gb2) or Gal(α1-4)Gal ceramide from marinesponge may be obtained, for example, from Dr T. Matsubara (Department ofChemistry, Kinki University, Kowakae. Japan).

VTs and Subunits

A simple method for purifying E. coli H30 verocytotoxin is as follows.The toxin, released from the cells by exposure to polymyxin B, issubjected to differential ammonium sulfate precipitation and sequentialchromatography on hydroxylapatite, chromatofocussing, Cibachron blue,and Sephadex G-100 columns. The purified toxin, 39 kDa by gel filtrationand having a pI of 6.72, resolves as a band which migrates at 32 kDa andanother band of less than 14 kDa which migrates with the buffer front onreducing SDS-PAGE. The purified preparation is relatively heat-stable,and has a specific activity of 3×10⁹ CD₅₀ units/mg protein in Verosells, and LD₅₀ values of 0.2, 9.0, and 40 g protein/kg in rabbits,rats, and mice, respectively. Antiserum to the toxin specificallyneutralizes H 30 VT, Shiga toxin, and VT activity from some clinicalisolates of VT⁺ E. coli but not that from a porcine edema diseasestrain. Verocytotoxin 2 (VT2) is purified from E. coli strain E32511using, as starting material, cells harvested from a Penassay brothculture incubated for 6 h at 37° C. in the presence of mitomycin C (0.2μg /ml). A crude extract of VT2, is obtained by polymyxin B treatment ofcell pellets, is purified using differential ammonium sulphateprecipitation, and sequential column chromatography. The purified toxinis estimated to have a pI of 6.5 by chromatofocusing and a molecularweight of 42 kDa by gel filtration; it has a specific activity of1.39×10⁶ CD₅₀ units/mg protein in Vero cells, and resolves as a majorband of Mr 35 kDa and another band of <14 kDa which migrates with thebuffer front on reducing SDS-PAGE. The purified toxin is not neutralizedby VT 1 antisera, and antisera prepared to this toxin in rabbits did notneutralize VT 1, but completely neutralized the activity of thehomologous toxin.

Recombinant Methods of Preparing VT's and Subunits

Recombinant VT1 is purified from pJLB28. VT2 from R82. and VT2c fromE32511. The recombinant E. coli strain pJLB28 is used as a source of VT1B subunit. High yields of the toxins or subunits (10-15 mg/3 liters ofbroth culture) are purified by a method involving polymyxin Bextraction, ultrafiltration, hydroxylapatite chromatography,chromatofocusing, and Cibacron Blue chromatography. VT2 is purified byvirtually the same method from an E. coli clinical isolate, strain E32511. The cistron encoding the B subunit of E. coli Shiga-like toxin I(SLT-I) is cloned under control of the tac promoter in the expressionvector pKK223-3 and the SLT-I B subunit is expressed constitutively in awild-type background and inducibly in a lacl^(q) background. E. coli TB1 lac pro rpsL ara thi □ 80d LacZ Δ MI5 hsdR is obtained from BethesdaResearch Laboratories (Gaithersburg, Md.). E. coli JM1O1 Δ lacc pro supEthi (F′ traD36 IacZ A MIS pro AB IacP) is obtained from Dr. J. D.Friesen (Department of Medical Genetics, University of Toronto, Toronto,Ontario, Canada). Plasmids pTZ18R and pKK223-3 are obtained fromPharmacia. Plasmid pJLB5 consists of a 3.0 kb Kpnl fragment ofbacteriophage H 19B DNA cloned in the Kpnl site of pUC18. To constructplasmid pJLB34, pJLB5 is cut at the BglII site and digested withnuclease Bal31. The ends are filled with Klenow fragment and dNTPs. Thefragment remaining after deletion is cleaved with EcoRI, and the piececarrying the SLT-I B cistron is purified by agarose-gel electrophoresis.The fragment is recovered from the gel and cloned into pUC18 cut withEcoRI and HindII. The EcoRl-HindIII fragment is cloned in M13mpl8 andits nucleotide sequence is determined. The B cistron coding sequence isrecovered from pJLB34 as a 1.1 kb Pstl fragment and was then cloned inthe PstI fragment and was the cloned in the PstI site of the polylinkerof pKK223-3. Clones with the correct orientation of insertion relativeto the tac promoter are identified by restriction-endonuclease analysis.One plasmid with the orientation is selected and designated pJLB120.pJLB120 is transformed into E. coli TBI for constitutive expression andinto E. coli JM101 for inducible expression. Bacteria are grown inL-broth or brain heart infusion broth (Difco Laboratories, Detroit,Mich.) supplemented as necessary with carbenicillin at 50 μg/ml and IPTG(Bethesda Research Laboratories) at 1 mM.

Expression of Toxins

For E. coli JM 101 (pJLB 120), an overnight culture is used to inoculatefresh L-broth supplemented with carbenicillin (50 pg/ml) and was grownto mid-exponential phase (A₆₀₀=0.3-0.6) at 37° C. with shaking at 300rev./min. IPTG is added to a final concentration of 1 mM. and incubationis continued with aeration. For E. coli TB I (pJLB 120), an overnightculture is used to inoculate fresh L-broth supplemented withcarbenicillin (50 μg/ml), and this is grown for 12-18 h at 37° C., withshaking at 300 rev./min. In both cases the culture is harvested and thepellet is washed once with PBS (0.15M-NaCl/10 mM sodium phosphatebuffer, pH 7.4) before extraction.

Polymyxin B extraction of Toxins

The washed pellet is resuspended in PBS containing 0.1 mg/ml polymyxin Bin one-quarter of the original culture volume and extracted aspreviously described. For purification, 18 h cultures of E. Coli TBI(pJLB 120) are extracted with polymyxin B, and the extracts areconcentrated 10-fold using a stirred-cell Amicon concentrator with aYm-5 membrane (Amicon Corp., Danvers, Mass., USA).

Quantification of Toxins

Periplasmic extracts of VT-producing clones, prepared by polymyxin Bextraction, are diluted as required and filtered onto nitrocellulosepaper in a slot-blot apparatus (Bio-Rad Laboratories). VT is detected byusing MAb 1 3C4 according to the Western-blot procedure described above.Blots are scanned with a Molecular Dynamics model 300A computingdensitometer. VT is quantified by comparison with a standard curvegenerated with purified B subunit protein.

Purification of Toxins

The concentrated polymyxin B extract are dialysed overnight against 50mM-Tris/HCl buffer, pH 7.4, and then applied to a DEAE-Sephacel column(1 cm×20 cm) equilibrated with 1 mM-Tris/HCL buffer, pH 7.4. Boundmaterial is eluted by using a linear gradient of 0-1M-NaCl in 50mM-Tris/HCl buffer, pH 7.4, and 5 ml fractions are collected. Fractionscontaining VT are identified, pooled and concentrated with Centriprep-3concentrators (Amicon Corp.). This pool is dialyzed overnight against 25mM-imidazole/HCl buffer, pH 7.4, and is applied to a column (1.5cm×20cm) of Polybuffer exchanger 94 (Pharmacia) equilibrated with thesame buffer. Elution is carried out with a degassed solution ofPolybuffer 74 (Pharmacia) diluted 1:8 with distilled water and adjustedto pH 4.0 with HCl (11column volumes). Fractions (5 ml) are collected,and the B subunit positive fractions are pooled and concentrated withCentriprep-3 (Amicon). Ampholytes are removed by Sephadex G-50gel-filtration.

HPLC Purification of Toxins

Approximately 1 mg (in 1 ml) of purified toxin or subunit is injectedinto a TSK-G2000SW HPLC gel filtration column previously equilibratedwith 50 mM Tris-buffered saline (TBS), pH 7.4, flow rate of 1.0 ml/mm.Peaks, measured by absorbance at λ=280 nm, are collected.

Toxin Subunit Separation

1 mg of toxin subunit is concentrated to 30-50 μl using a Centricon 30concentrator (Amicon). 1 ml of a subunit dissociating solution (6 Murea, 0.1 M NaCl, 0.1 M propionic acid, pH 4, is added dropwise, and thetoxin is incubated without stirring at 4° C. for 1 h. The solution isthen separated by HPLC gel filtration (as above) after previous columnequilibration with the dissociating solution. Peaks, measured byabsorbance at λ=280 nm, are collected.

EXAMPLE 42 Gangliosides Shed from Tumor Cells: Isolation from Tumor CellSupernatants Collection of Tumor Cell Supernatant

Tumor cells are cultured in 25 ml of no serum-low protein medium (NSLP)in an 80cm²flask for 1-5 days. Cells are harvested by centrifugation at400 g for 10 mm, and the supernatant is concentrated 10-fold at 4° C. inan Amicon stirred cell with a 10-kDa cutoff ultrafilter. Concentratedsupernatant and NSLP concentrated under the same conditions are storedat −20° C., and passed through a 0.1-um sterile membrane filter.

Metabolic Labeling of Gangliosides in Tumor Cell Supernatant

Tumor cells (1×10⁵/ml) are cultured in 10 ml of NSLP for 2 days. Afterthree washes with fresh medium, cells are transferred into 10 ml of NSLPcontaining 1 μCi/ml D-[1-¹⁴C]GlcNH₂-HCl (50 mCi/mmol (ICN Biomedicals,St. Laurent, Quebec, Canada) and 1 μCi/ml of D-[1-¹⁴C]Gal (56 mCi/mmol;Amersham) to label gangliosides. After 24 hr, cells are washed withmedium three times to remove unincorporated sugars, then cultured for anadditional 24-48 hr in fresh medium, before harvesting by centrifugationat 400 g. Radioactivity in the tumor cell supernatant and cells isquantitated by liquid scintillation counting. The supernatant isclarified by centrifugation at 15,000 g for 10 mm, then concentrated10-fold using a Speedvac concentrator, before being analyzed by gelfiltration chromatography.

Gel Filtration Chromatography of ¹⁴C-Labeled Tumor Cell Supematant onSepharose 2B-300

Concentrated ¹⁴C-labeled tumor cell supernatant is chromatographed on aSepharose 2B-300 column (5 ml bed volume; Sigma Chemical Go, St Louis,Mo.), equilibrated with Tris-buffered saline (TBS; 50 mM Tris-HCl in0.15 M NaCl, pH 7.4). The column is eluted at a flow rate of 0.2 ml/minat 22° C., and 2001 μl fractions are collected and counted for ¹⁴C.Dipalmitoylphosphatidylcholine liposomes and sodium azide are used asstandards to calibrate the void and included volume of the column,respectively.

Gel Filtration FPLC of 'P-Labeled Tumor Cell Supernatant on Superose

FPLC is carried out on a Superose 6 column (1×30 cm; Pharmacia, Dorval,Quebec, Canada) linked to a Gilson HPLC system and a Gilson iliBultraviolet flow detector. The column is calibrated with a series ofstandard proteins of known molecular mass, ranging from □-galactosidase(465 kDa) to □-lactoglobulin (36.8 kDa) (Pharmacia, High MolecularWeight Gel Filtration Calibration kit). The void volume and includedvolume are determined using Blue Dextran (2000 kDa) and sodium azide,respectively. ³H-Labeled bovine brain gangliosides and [¹⁴C]Galdissolved in NSLP or TBS are also used as standards. Concentrated YAC-1supernatant is eluted through the column at 22° C. with TBS at a flowrate of 0.5 ml/min. Fractions (0.5 ml) are collected and counted for¹⁴C.

EXAMPLE 43 Assessment of SAg and VT Binding to Glycosphingolipids by TLCOverlay

Glycolipids (dissolved in chloroform/methanol (2:1 v/v), are applied toa TLC plate and separated in choroform/methanol/water (65:25:4, v/v).Toxin binding is determined using known methods. Briefly, afterseparation of the glycolipids, the plate is air dried, incubatedovernight at 37° C. in a solution of 1% (m/v) gelatin in 50 mM Tris/HCL,150 mM NaCl, pH 7.4 (buffer A). The plate is washed in buffer A andincubated successively with VT 1 (0.07 μg/ml in buffer A) followed bymonoclonal antibody PHI (1.5 pg/ml in buffer A), and finally with goatantimouse IgG horseradish peroxidase conjugate (diluted 1:2000 in bufferA). Toxin binding is visualized using 4-chloro-1-naphthol. An equivalentplate is run and treated with 3% (m/v) orcinol spray in 3 M H₂SO₄ tovisualize carbohydrate and ensure equal concentrations.

Alternate Microtitre Plate Binding Assay

Quantification of toxin binding to various glycoconjugates is performedusing published methods. A methanolic solution [100 pl containingglycolipid (300 nmol). phosphatidylcholine (0.5 μg) and cholesterol(0.25 μg)] is added to microplate wells and the methanol is allowed toevaporate overnight at room temperature. The wells are blocked with 2%(m/v) BSA in buffer A (200 μl/well) for 2 h at room temperature andsubsequently washed once with buffer A containing 0.1% BSA (BSA/bufferA). 100 μl aliquots of dilutions of [¹²⁵I]-VT-1 in BSA/buffer A areadded to the wells and incubated for 2 h at room temperature. The wellsare washed five times with BSA/buffer A. excised and the radioactivityis measured in a γ counter. Scatchard analysis was performed using theLIGAND program.

EXAMPLE 44 Methods of Induction and Assessment of Apoptosis & Inhibitionof Protein Synthesis.

Tumor cells (5×10⁵cells/ml) are cultivated at 37° C. in 96-wellround-bottomed microtiter plates (Becton Dickinson) in 200 μlleucine-depleted RPMI (Eurobio, France) containing 1 μCi of [³H]leucine. with or without 10 ng/ml VT. After 18 hrs. cells are harvestedon class fiber filters, and radioactivity incorporated in proteinsmeasured in a scintillation counter.

Ultrastructural Analysis of VT-Treated Astrocytoma Cells

Cells are cultivated on a transferable 9 mm cylcopore membrane (0.45 μmpore size. Falcon) to form a confluent monolayer and are incubated at37° C. with VTI (10 ng/ml). Cells are fixed at room temperature byaddition of 1.6% glutaraldehyde to the wells and then incubated in 0.066M Sorensen buffer (pH 7.4) containing 1.5% glutaraldehyde for 1 h at 4°C. After 2 h of washing with 0.1 M phosphate buffer, cells arepost-fixed in 2% osmium tetroxide in the same buffer. After dehydrationin graded ethanols and propylene oxide, Epon embedding, thin sectioningand uranyl-lead counterstaining on grids are performed. Thin sectionsare examined in a Philips EM 400 electron microscope and ultrastructuralfeatures of apoptosis are analyzed

Flow Cytometry

Apoptosis of astrocytoma cells, incubated with 10 ng/ml of VT1 for 24-36hrs in the presence of 10% bovine fetal serum is analyzed on an EpicsProfile Analyzer (Coulter Electronics. Pathology. University of Toronto)according to known procedures. After treatment, cells are trypsinizedand the 200×g centrifuged cell pellet is suspended in 1 ml of hypotonicfluorochrome solution of 50 μg/ml propidium iodide (Sigma) and stainedfor 30 min at 4° C. To remove RNA prior to staining. cells are treatedwith 100 μl of 200 μg/ml solution of DNase-free RNase A at 37° C. for 30min. Cell cycle distribution is determined using manual gating. Flowcytrometric quantitation of apoptotic cells within the propidiumiodide-stained population is performed as described. Debris and deadcells are excluded on the basis of their forward and sidelight-scattering properties. Astrocytoma cells grown simultaneously inthe absence of VT1 serve as controls.

DNA Fragmentation Assays Cells:

Tumor cells are incubated in RPMI 1640 medium alone or in the presenceof intact VT or VT-B. After 18-h culture, cells are counted andviability assessed by trypan blue exclusion. Cells are then centrifugedand washed twice with saline buffer. The pellets are lysed by incubationfor 1 h at 50° C. in 10 mM EDTA, 200 mM NaCl, 0.1 mg/ml proteinase K,0.5% (w/v) SDS, and 50 Mm Tris-HCL, pH 8. The DNA is extracted withphenol, chloroform:isoamylalcohol (24:1), and then ethanol precipitated.Unfragmented DNA is discarded, and 0.1 volume of 3 M sodium acetate, pH7.2, is added to the supenatant which is left at −80° C. overnight. Theprecipitate containing fragmented DNA is centrifuged (1300 g, 30 mm) anddried under vacuum. DNA derived from 5×10⁶ cells is then resuspended in20 μl RNAse buffer containing 0.5 μg/ml DNAse-free RNAse (Sigma), 15 mMNaCl, and 10 mM Tris-HCL, pH 7.5. and incubated at 50° C. for 1 h;Electrophoresis is carried out at 70V in 2% agarose gel containing 0.1μg/ml ethidium bromide in a buffer containing 2 mM EDTA, 80 mMTris-phosphate. pH 8. After electrophoresis. gels are examined under UV.Phage DNA from bacteriophage λ and □ digested by HindIII and HaeIII,respectively, provide molecular weight standards.

Nuclear Staining with Propidium Iodide

SF-539 cells grown on the cover slips overnight are incubated at 37° C.with VT-12B subunit (50 , g/ml) for 1.5 hrs or 10 hrs and fixed (with 1%paraformaldehyde for 3 minutes). permeabilized with 0.1% Triton X in 100mM PBS for 5 min, and stained with 5 μg/ml propidium iodide (Sigma).After extensive wash with 50 mM PBS, the fixed cells are mounted withDABCO (1,4-diazabicyclo-octane (Sigma), and nuclear staining is observedunder incident UV illumination.

Proliferation Assay

Approximately 1-5×10⁴ cells are added to 24-well plates and incubated ina-MEM in 5% CO₂ at 37° C. After 24 hr, the growth medium is replacedwith medium containing various concentrations of the holotoxin VT1(0.0.1.5.50, 100 ng/ml). The treated astrocytoma cell lines andendothehal cells are trypsinized and counted at intervals throughout thegrowth curve. Cell viability is assessed by trypan blue dye exclusion.Cell counts are plotted against time for the various concentrations ofVT1 and B subunit. For each time point analyzed, the wells are set-up intriplicate.

For selected cell lines, the B subunit of VT1, VT2, and VT2c is addedalone to the astrocytoma cells at same concentrations listed above. Asingle dose of VT 1. VT2. and VT2c is added to confluent astrocytomacells in microplate wells. Cell survival at 72 hr is monitored bystaining with 0.1% crystal violet, and measuring the optical density at590 nm using a Dynatek microtiter plate reader.

EXAMPLE 45 Multidrug Resistant Cells: Culture and Preparation

MCF-7-wt and MCF-7-AdR (adriamycin-resistant) cells are obtained fromDrs. K. H. Cowan and M. B. Goldsmith, National Cancer Institute. Cellsare maintained in RPMI 1640 medium containing 10% FBS (v/v), 50 units/mlpenicillin, 50 μg/ml streptomycin, and 584 mg/liter L-glutamine. KB-3-1human oral epidermoid carcinoma cells (parent, drug-sensitive) andKB-V-1 cells (highly MDR) and subclones are obtained from the NationalCancer Institute). Cells are grown in high glucose (4.5 g/liter)Dulbecco's modified Eagle's medium containing 10% FBS and othercomponents described above. The KB-V-1 cell line is maintained withvinblastine (1.0 μg/ml) in the medium. NIH:OVCAR-3 cells (human ovarianadenocarcinoma, drug-resistant) are obtained from the American TypeCulture Collection and grown in RPMI 1640 medium containing insulin (10μg/ml), 10% PBS, and other components listed above. All cells arecultured in a humidified, 6.5% CO₂ atmosphere, tissue culture incubator.Cells are subcultured once a week using 0.05% trypsin and 0.53 mM EDTAsolution.

Lipid Mass Analysis

Cell lipids are analyzed by TLC separation and charring of thechromatogram. Briefly, total cellular lipids are extracted and equalaliquots (by weight) from each sample are spotted on TLC plates. Platesare developed in the desired solvent system (see below), air-dried for 1h, and sprayed using a 35% solution of sulfuric acid in water (v/v). Thelipids are charred by heating in an oven at 180° C. for 30 mm, andresulting black bands are visualized.

Cell Radiolabeling and Analysis of Sphingolipids

MCF-7 cells grown in medium containing 10% FBS, are switched toserum-free medium containing 0.1% fatty acid-free BSA. Cell lipids areradiolabeled by incubating cells with [³H]serine (2.0 μCi/ml),[³H]palmitic acid (1.0 μCi/ml, or [³H]galactose 1.0 μCi/ml) for theindicated times. In some instances, cells are radiolabeled in mediumcontaining 5% PBS. Cells are then rinsed twice with PBS, and 2 ml ofice-cold methanol containing 2% acetic acid is added. The cells arescraped free, transferred to glass test tubes (13×100 mm), and lipidsare extracted, by the addition of chloroform (2 ml) followed by water (2ml). The resulting organic lower phase is evaporated under a stream ofnitrogen. Lipids are resuspended in 1001 μl of chloroform′methanol (1:1,v/v) and aliquots are applied to TLC plates. When using [³H]galactose,radiolabeled cells are washed twice with PBS, transferred to glass tubeswith methanol (2 ml, and glucosylceramides and gangliosides (2.5 pg ofeach) are added to aid recovery. Lipids are extracted by the addition ofwater (2 ml; and 2 ml of chloroform (three times consecutively). Thepooled organic lower phase is treated as above. Lipid analysis iscarried out by various TLC separations using solvent system I,chloroform/methanol/ammonium hydroxide (65:25:5, v/v); solvent systemII, chloroform/methanol/ammonium hydroxide (40:10:1, v/v), solventsystem III, chloroform/methanol/water (60:40:8, v/v), or solvent systemIV, chloroform/methanol/acetic acid/water (50:30:7:4, v/v). Fordetermination of ceramides. an aliquot of the chloroform-soluble lipidsis base-hydrolyzed in 0.1 N KOH in methanol for 1 h at 37 ° C.; lipidsare re-extracted and separated using solvent system V hexane/diethylether/formic acid (60:40: 1, v/v). Galactosyl- and glucosyl-ceramidesare separated using solvent system VI, chloroform/methanol/water(60:25:4, v/v). This separation is performed on TLC plates that arepre-run in 2.5% borax in methanol/water (1:1) and heated at 110° C.prior to use.

Radiochromatograms are sprayed with EN³HANCE and exposed for 3-7 daysfor autoradiography. TLC areas, aligned with hands on theautoradiographs or with iodine-stained commercial lipid standards arescraped from the plate. Water (0.5 ml) is added to the plate scrapings,followed by 4.5 ml of EcoLume counting fluid, and the samples arequantitated by liquid scintillation spectrometry.

Purification of Glycosylceramides

The compounds, extracted with total lipids from MCF-7-AdrR cells, areresolved from other lipids on preparative TLC using silica gel H platesdeveloped in solvent system II. The appropriate region of the TLC plateis then scraped into test tubes, and lipids are extracted withchloroform/methanol/acetic acid/water (50:25:1:2, v/v). The samples arecentrifuged, and the solvent transferred to new glass tubes andevaporated to dryness under nitrogen.

Fast-Atom Bombardment/Mass Spectrometry of TLC-isolated Lipid-FAB/MSspectra are acquired using a VG 70 SEQ tandem hybrid instrument of EBqQgeometry (VG analytical, Altrincham, UK.). The instrument is equippedwith a standard unheated VG FAB ion source and a standard saddle-fieldgun (Ion Tech Ltd., Middlesex, UK) that produces a beam of xenon atomsat 8 kV and 1 mA. The mass spectrometer is adjusted to a resolving powerof 1000, and spectra are obtained at 8 kV using a scan speed of 10s/decade. 2-Hydroxyethyl disulfide is used as matrix in the positiveFAB/MS, and triethanolamine is used as a matrix in the negative FAB/MS.Negative FAB and positive FAB give different values for the samecompounds, due to charge (proton content) differences.

EXAMPLE 46 Incubation of Tumor Cells with Hydroxy Fatty Acids forSelective Synthesis of Galactosphingolipids and Lipid Analysis

Tumor cells on filters are incubated for 1 hr at 37° C. in the presenceof labeled and unlabelled [³H]Cer(C₆[D-20H]). After the incubation,lipids are extracted from the cells and the combined incubation mediaand analyzed Lipids are extracted from cells and media by a two-phaseextraction. The upper phase contains 20 mM acetic acid and (forradiolabeled lipids) 120 mM KCl. After a chloroform wash. which is addedto the lower phase, lipids remaining in the upper phase GalCer arecollected on SepPak C18 cartridges (Waters, Milford, Mass.) from whichlipids are eluted with chloroform/methanol/water 1:22:0.1) and methanol.The organic (lower) phase is dried under N₂, and the lipids are appliedto TLC plates that were dipped in 2.5% boric acid in methanol, dried,and activated by heating at 110° C. for 30 mm. They are developed in twodimensions:

-   I. chloroform/methanol/25%NH₄OH/water (65:35:4:4. v/v); and-   II. chloroforrm/acetone/methanol, acetic acid/water (50:20:10:10:5.    v/v).    Fluorescent spots are detected under UV. scraped from the TLC plates    and the fluorescent lipid analogs are extracted from the silica in 2    ml chloroform/methanol/20 mM acetic acid (1:2:2:1 v/v) for 30 mm.    After pelleting the silica for 10 min at 1,500 rpm fluorescence in    the supernatants is quantified in a fluorimeter (Kontron. Zorich,    Switzerland). Radiolabeled spots are detected by fluorography after    dipping the TLC plates in 0.4% PPO in 2-methylnaphthalene with 10%    xylene. Preflashed film (Kodak X-Omat S) is exposed to the TLC    plates for 3 d at −80° C. The radioactive spots are scraped from the    plates, and the radioactivity is quantified by liquid scintillation    counting in 0.3 ml Solulyte (J.T. Baker ChemicaL Deventer, The    Netherlands) and 3 ml of Ultinsa Gold (Packard Instruments. Downers    Grove. Ill.).

EXAMPLE 47 Conjugation of Proteins to Lipoproteins

The preferred method for coupling superantigens to lipoproteins is touse 10 mM solution of sodium periodate for oxidation of the carbohydratein the lipoprotein. This will also cleave c-c bonds in the sugars withadjacent hydroxyls and oxidize them to reactive aldehydes. Superantigensform Schiff base linkages with the aldehyde modified sugar groups underalkaline conditions. the aldehyde modified sugar is then coupled to theamine containing superantigen peptide or polypeptide. The oxidation isfollowed by reductive amination using sodium cyanoborohydride to reducethe labile Schiff base between the aldehyde on the carbohydrate and theamine on the superantigen to form stable secondary amine covalentlinkages. An alternative procedure is to periodate oxidize thelipoprotein as above to create reactive aldehyde groups.Heterobifunctional cross-linking agent such as4-(4-N-Maleimidophenyl)butryric acid hydrazide (MPBH)4-(4-N-Maleimidophenyl)buryric acid hydrazide (MPBH), and4-(N-Maleimidomethyl)cyclohexane-1-carboxyl-hydrazide (M2C2H) whichcontain a carbonyl-reactive hydrazide group on one end and asulfhydryl-reactive maleimide on the other are preferred. The hydrazidereacts specifically with aldehyde functional groups to create ahydrazone linkage a type of Schiff base. To stabilize the bond betweenthe hydrazide and aldehyde, the hydrazone is reacted with sodiumcyanoborohydride to reduce the double bond and form a secure covalentlinkage. The cross-bridge between the two functional ends provides along, 17.9-A spacer. These agents couple to periodate-oxidized aldehydeson the lipoportein carbohydrate via the hydrazine and to sulfhydrylgroups on the superantigen via sulfhydryl reactive maleimide group.Superantigens without reactive sulfhydryl groups are first thiolatedwith SATA or Trout's reagent before addition to the reactive maleide. Asulfhydryl-containing protein or molecule is is bound via the maleimideend of MPBH and the derivative purified by gel filtration to removeexcess reactants, and then mixed with a lipoprotein (that had beenpreviously oxidized to provide aldehyde residues) to effect the finalconjugation. The opposite approach e.g., modification of theglycoprotein first, purification, and subsequent mixing with asulfhydryl-containing molecule is also acceptable. With this secondoption, however, the purification step should be done quickly to preventextensive hydrolysis of the maleimide group. (See Hermanson G TBioconjugate Techniques Academic Press, San Diego Calif., 1996)

Protocol for Periodate Oxidation.

-   1. Periodate-oxidize a liposome suspension containing glycolipid    components according to Section 2. Adjust the concentration of total    lipid to about 5 mg/ml.-   2. Dissolve the protein to be coupled in 20 mM sodium borate, 0.15 M    NaCl, pH 8.4, at a concentration of at least 10 mg/ml.-   3. Add 0.5 ml of protein solution to each milliliter of lipoprotein    suspension with stirring.-   4. Incubate for 2 h at room temperature to form Schiff base    interactions between the aldehydes on the lipoprotein and the amines    on the protein molecules.-   5. In a fume hood, dissolve 125 mg of sodium cyanoborohydride in 1    ml water (makes a 2 M solution). This solution may be allowed to sit    for 30 mm to eliminate most of the hydrogen-bubble evolution that    could affect the lipoprotein suspension.-   6. Add 10 ul of the cyanoborohydride solution to each milliliter of    the lipoprotein reaction.-   7. React overnight at 4° C.-   8. Remove unconjugated protein and excess cyanoborohydride by gel    filtration using a column of Sephadex G-50 or G-75.

EXAMPLE 48 Isolation of Lipoproteins

Human LDL is isolated by sequential ultracentrifugation (d 1.019-1.063g/ml) from freshly drawn, citrated normolipidemic human plasma to whichEDTA 0.1 mmol/liter is added. Freshly obtained plasma is subjected todifferential ultracentrifugation to isolate the desired lipoproteinfractions . Typically, the following density fractions were isolated: 1)d<1.02. to remove VLDL and IDL; 2) d=1.02-1.05, to obtain LDL; 3)d=1.05-1.08, to obtain Lp(a); and 4) d=1.08-1.21, to obtain Lp(a) andHDL. The Lp(a)-containing density fractions were subjected to gelfiltration chromatography on a Bio-Gel A-15 m column (2.5×90 cm). Thiscolumn was eluted with 1.0 M NaCl, 10 mM Tris, 10 mM NaN3, 1 mM EDTA, pH7.4, and was continuously monitored at 280 nm. The LDL- andHDL-containing density fractions are also subjected to gel filtrationchromatography to remove any contaminating species and for uniformity ofsample preparation. They are further dialyzed against 0.01 M sodiumphosphate pH 7.4, containing 0.15 M sodium chloride and 0.01% EDTA,sterilized on 0.2-um Millipore membrane, and stored at 4° C. undernitrogen (up to 3 weeks).

Lipoprotein (a) (Lp(a))

Lp(a) is prepared from fresh human plasma by flotation centrifugationfollowed by affinity chromatography on lysine-Sepharose and CsCI densitygradient centrifugation as described. Lipoprotein preparations aredialyzed against 0.15 M sodium chloride containing 0.01% EDTA at 0.01%sodium azide, filter sterilized (0.45 pm) and stored at 4° C. in vialsfilled to allow no air space. No contamination of the preparations byplasminogen is detected by either Coomassie Blue staining of sodiumdodecyl sulfate (SDS) gels or by treatment with streptokinase andmeasuring plasmin activity with a chromogenic substrate. S2251. Thesensitivities of these assays excluded plasminogen contamination of >1%and >0.4% respectively. Lp (a)-free LDL, HDL and acetylated LDL areprepared as previously described. The LDL contained no apoA-1 and theHDL contained no detectable apoB-100. The apoprotein composition isverified by SDS polyacrylamide gel elecrophoresis.

Lysine-Sepharose Chromatography

Lipoprotein (a) has an affinity for lysine-Sepharose by virtue of lysinebinding kringle 4 domain(s) located on apo(a). The most important domainappears to be kringle 4₃₇, which has the greatest homology to kringle 4of plasminogen, although there may be other kringles with lesseraffinity for lysine which also contribute to the interaction of Lp(a)with lysine-Sepharose. Plasminogen and Lp(a) have similar affinities forlysineSepharose; however, Lp(a) species with different apo(a) isoformsmay have affinities that are significantly greater or weaker than thatof plasminogen. The buffer of choice in the isolation of plasminogenfrom plasma by lysine-Sepharose affinity chromatography has been 0.1 Mphosphate buffer, pH 7.4. When the same buffer system is used in thechromatography of Lp(a), not all the lipoprotein is found to bind to thelysine-Sepharose i.e., approximately 80% of Lp(a) contained in theplasma had the capacity to interact with lysine-Sepharose. Thepercentage of Lp(a) binding to lysine-Sepharose is increased by loweringthe ionic strength of the buffer medium. Lipoprotein (a) species withlarge apo(a) isoforms tend to self-associate in the cold therefore it isbest to perform the chromatographic isolation at room temperature.

Preparation of Lysine-Sepharose 4B

Packed Sepharose 4B (250 ml) is washed with 8 liters of water on acoarse sintered glass funnel and activated with 25 g CNBr dissolved in50 ml acetonitrile. The reaction is carried out in a well-ventilatedhood, on ice, and the pH is maintained with 6 N NaOH at pH 11. Afterapproximately 15 to 30 mm, the activated Sepharose 4B is washed with 8liters of 0.1 M NaHCO₃ pH 8.1. The agarose is then packed by filtration,diluted with 250 ml of 0.1 M NaHCO₃ pH 8.1, containing 50 g lysine, andstirred gently overnight at 4°. The freshly conjugated lysine-Sepharoseis then washed with 6 to 10 liters of 1 mM HCl followed by 8 liters of0.1 M NaHCO3, pH 8.1, and an aliquot is saved for determination of theconcentration of immobilized lysine residues using the method of Wilkieand Landry. The concentration of coupled lysine varies from 15 to 25umol per milliliter packed gel

Chromatography

Bio-Rad (Richmond, Calif.) Econo-Pac columns (1×12 cm) are packed with 5ml lysine—Sepharose which is preequilibrated with column buffer (e.g.,0.1 M phosphate, 0.01% NaN₂, pH 7.4). A porous polymer filter is placedon top of the lysine-Sepharose gel bed to prevent the column fromrunning dry. Plasma samples smaller than 3 ml are applied to the columnand allowed to run through by gravity at room temperature. Largervolumes (up to 50 ml) should be applied with a pump or by gravity, butat flow rates that should not exceed 20 ml/cm²/hr. The samples arewashed into the column with four 0.5-ml aliquots of column buffer to befollowed with four 0.5 ml aliquots, before Lp(a) is eluted withe0.2<EACAin 10 mM phosphate, pH 7.4. One milliliter aliquots are applied at atime, and 1 ml fractions are collected in separate tubes.Liprotein(a)and plasminogen-containing fractions (tubes 4 through 10)are located by their absorbance at 280 nm. The volume of applied plasmadepends on the Lp(a) content and on the sensitivity of the absorbancemonitor that is part of the density gradient fractionating system.

Density Gradient Centrifuigation of Lp(a)

Place 5 ml of 20% (w/w) NaBr into a SW-40 ultracentrifuge tube(ultraclear). Carefully layer the eluate from the lysine-Sepharosecolumn (up to 8 ml) on top of the NaBr solution and, if necessary, topoff the tube with 0.2 M EACA, 10 mM phosphate, pH 7.4. Place the tubesin the bucket of the swinging-bucket rotor and centrifuge 64 hr at39,000 rpm and 20°. After centrifugation is completed, the tubes arecarefully removed from the buckets and placed in the density gradientfractionating system. The tubes are pierced at the bottom, and thegradient is pushed out the top at a flow rate of 1 ml/min with a densefluorocarbon oil, Fluorinert FC-40 (ISCO), that has a density of 1.85g/ml. The chart speed is 1 cm/min, and the fraction collector is set to0.5 ml/tube. The gradient is monitored at 280 nm, and the sensitivity ofthe chart recorder is adjusted according to the Lp(a) content of theeluate. Densities of the various fractions are measured with a densitymeter by established techniques.

Isolation of Apolipoproteins B-48 and B-100

The following density gradient ultracentrifugation procedure forisolating subfractions of triglyceride-rich lipoproteins is suitable forSDS-PAGE on both slab and rod gels. Plasma is recovered by low speedcentrifugation (1750 g, 20 min, 10). To minimize proteolytic degradationof apo B, 1.0 ul/ml plasma phenylmethylsulfonyl fluoride (PMSF, Sigma,St. Louis, Mo.), 10 MM dissolved in 2-propanol, and 5 ul/ml plasmaaprotinin (Trasylol, Bayer, Leverkusen, (Germany), 1400 ug/liter, areadded. Subsequently 140.4 mg solid NaCl is added per 1.0 ml plasma toincrease the density to 1.10 kg/liter. Normally, a total volume of 4.0ml of the d 1.10 kg/liter plasma is put in the bottom of a 13.4-mlpolyallomer ultracentrifuge tube (Ultra-Clear, Beckman Instruments, PaloAlto, Calif.). Alternatively, 3.0 ml plasma can be mixed with 1.5 mIt)1.42 kg/liter NaUr, from which 4.0 ml is transferred to theubracentrifuge tube. For the rod gel method, two such tubes are requiredto obtain enough material from each sample. For the slab gel method, 1.0ml plasma is sufficient. In the latter case, a 1.0 ml portion of 1.10kg/liter plasma can be mixed with 3.0 ml of 1.10 kg/liter NaCl in thetube. A density gradient consisting of 3.0 ml each of 1.065, 1.020, and1.006 kg/liter NaCl solutions is then sequentially layered on top of theplasma. Ultracentrifugation is performed in a SW4O Ti swinging bucketrotor (Beckman) at 40,000 rpm and 15° (Beckman L8-55 ultracentrifuge).Consecutive runs calculated to float Svedberg flotation rate (Sf) >400(32 min), SI 60-400 (3 hr 28 mm), and Sf 20-60 (14-16 hr) particles aremade. After each centrifugation, the top 0.5 ml of the gradientcontaining the respective lipoprotein subclasses is aspirated, and 0.5ml of density 1.006 kg/liter salt solution is used to refill the tubebefore the next run. The Sfl 12-20 fraction is recovered after the lastultracentrifugal run by slicing the tube 29 mm from the top after the Sf20-60 lipoproteins have been aspirated. All salt solutions should beadjusted to pH 7.4 and contain 0.02% (w/v) NaN3 and 0.01% Na2EDTA. Thismethod yields lipoprotein preparations almost completely devoid ofplasma albumin.

EXAMPLE 49 Preparation & Isolation of Oxidized LDL (oxyLDL

Oxidized LDL (oxyLDL)

Native LDL (200 ug protein/ml) is oxidized by exposure to 5 uM CuSO4 for24 h at 25° C. and the degree of oxidation is assessed by the increaseof mobility on 1% agarose gel (1.3-1.5 versus native LDL) and theformation of thiobarbituric acid-reactive substances (3.41+/−0.8mmol/L). Oxidation is terminated by refrigeration. Differentpreparations of oxyLDL display similar electrophoretic mobilities. Forcomparison, commercially available preparations of native andcopper-oxidized LDLs (Sigma Chemical Co., St. Louis, Mo. and BiomedicalTechnologies, Inc. Stoughton, Mass. respectively) are used. The level ofLDL oxidation is evaluated by monitoring the formation of lipidhydroperoxides, using the FOX-2 procedure and thiobarbituricacid-reactive substances (TBARS)). The relative electrophoretic mobilityis evaluated on Hydragel (Sebia, Paris, France) and the level oftrinitrobenzenesulfonic acid-reactive amino groups was determined

The formation of thiobarbituric acid-reactive substances is 17.8nanomoles of malondialdehyde/mg protein using an oxyLDL preparation withrelative electrophoretic mobility of 1.4.

Methods for Measurement of Low-Density Lipoprotein Oxidation

Oxidation of LDL in vitro is accompanied by characteristic changes ofchemical, physicochemical, and biological properties, and a variety ofmethods may therefore be used for determining the extent and/or rate ofoxidation of LDL. They include measurement of the increase ofthiobarbituric acid-reactive substances (TBARS), total lipidhydroperoxides defmed lipid hydroperoxides, hydroxy and hydroperoxyfatty acids, conjugated dienes, oxysterols, lysophosphatides, aldehydesand fluorescent chromophores as well as measurements of thedisappearance of endogenous antioxidants and polyunsaturated fattyacids, and oxygen uptake. The apolipoprotein B (apoB) becomesprogressively altered during oxidation; its loss of reactive aminogroups and fragmentation to smaller peptides is determined and used asan index of oxidative modification. The net increase of the negativesurface charge of the whole LDL particle is analyzed as relativeelectrophoretic mobility (REM) by agarose gel electrophoresis. Thebiological assays used most frequently for assessment of the extent ofoxidative modification are the rate of uptake of LDL by culturedmacrophages and its cytotoxicity toward cultured cells. Immunologicalassays such as enzyme-linked immunosorbent assay (ELISA) andradioimmunoassay (RIA) employing polyclonal or monoclonal antibodiesrecognizing certain modifications in apoB characteristic for oxidativemodification are employed. The epitopes produced by covalent binding ofmalonaldehyde or 4-hydroxynonenal are of particular interest. Nuclearmagnetic resonance (NMR), electron spin resonance (ESR), circulardichrorism (CD), and fluorescence polarization have also been applied tostudy certain aspects of LDL oxidation. Simple methods, such as themeasure of TBARS, conjugated dienes, or fluorescence are preferred. Mostcharacterize oxidized LDL by at least two independent measurements, forexample, TBARS or REM and macrophage uptake, antioxidants and conjugateddienes.

From kinetic experiments one can conclude that both cell-mediatedoxidation of LDL and oxidation in the absence of cells catalyzed by Cu²⁺ions proceed in three consecutive time phases: lag phase, propagationphase, and decomposition phase. 1)during the lag phase the LDL becomesdepleted of antioxidants, and during this period only minimal lipidperoxidation occurs in LDL, as shown by measuring polyunsaturated fattyacids (PUFAs), TBARS, lipid hydroperoxides, fluorescence, and conjugateddienes. When LDL is depleted of its antioxidants, the rate of lipidperoxidation rapidly accelerates and a lipid peroxide maximum is reachedafter about 70-80% of the LDL, PUFAs are oxidized. Thereafter, theperoxide content of LDL, starts to decrease again because ofdecomposition reactions. During the lag and propagation phases the timeprofile for TBARS, fluorescence at 430 nm, lipid peroxides, dienes, andREM are very similar and only after the peroxide maximum do thedifferent indices separate and follow different kinetics. This alsoindicates that all the methods will give equivalent results for thesusceptibility of LDL to oxidation as measured by the duration of thelag time.

Preparation of Low-Density Lipoproteins for Oxidation

Isolation of Low Density Lipoproteins

After overnight fasting blood samples are withdrawn by venipuncture andcollected by free flow of blood into plastic tubes containing theappropriate volume of an aqueous solution of 10% EDTA (w/v) (disodiumsalt, pH 7.4) to obtain a final blood concentration of 0.1% EDTA (wlv).EDTA serves as anticoagulant and antioxidant. Blood is centrifuged at1000 g for 10 mm; the supernatant is then centrifuged at 10° C. and 1000g for 5 min, followed by centrifugation at 15,000 g for 10 min. Thisprocedure removes all cellular debris, and a completely clear plasma isobtained. Generally plasma is not stored but is used the same day forLDL. isolation. The most common method for isolation of LDL is atwo-step sequential ultracentrifugation with a total run duration ofabout 48 hr. LDL is prepared for oxidation experiments by a single 20-hrrun with a discontinuous density gradient. Plasma (up to 4 ml) adjustedwith solid KBr to a density of 1.22 g/liter is layered on the bottom ofa centrifuge tube (Beckman polyallomer tubes, total volume 13.2 ml) andthen overlaid by KBr density solutions of 1.08 (3 ml), 1.05 (3 ml), and1.00 g/liter (to fill the tube) containing 1 g/liter EDTA (pH 7.4). Alldensity solutions are purged with nitrogen before use. The tubes arecentrifuged in a Beckman SW 41 Ti rotor at 40,000 rpm at 10° for 20 hr.After centrifugation the main lipoproteins very low-density lipoproteins(VLDL), LDL, and high-density lipoproteins (HDL) are well separated fromeach other, and the LDL band characterized by the yellow color due tothe endogenous b-carotene, is collected by aspiration with a syringe andtransferred into a polycarbonate tube.

Next, the cholesterol content of the isolated LDL sample is determinedwith the CHOD-PAP enzymatic test kit (Boehringer, Mannheimn, Germany).When 4 ml normolipidemic plasma is centrifuged, the final LDL stocksolution harvested from the ultracentrifugation has a concentration oftotal cholesterol of about 1.6 to 2.2 mg/ml. Based on the knowncomposition of LDL the total cholesterol values can be converted to LDLmass per milliliter (multiply cholesterol by the factor 3.16) or LDLprotein per milliliter (multiply total cholesterol by the factor 0.63).It is also possible to determine the LDL concentration by proteinmeasurement. Next EDTA is from the LDL stock solution and the oxidationis conducted immediately after isolation of LDL. For storage the LDLstock solution is sterile filtered through a 0.3 um filter adapted to asyringe into a sterile, evacuated glass vial and subsequently purgedwith nitrogen (Techne ViaL Mallinckrodt-Diagnostica, Holland, orBehring, Marburg, Germany).

Removal of EDTA

Removal of EDTA and salt from the density gradient from the LDL stocksolution is conducted with prepacked columns (Econo-Pac 10DG, Bio-Rad,Richmond, Calif.) filled with Bio-Gel P6 as desalting gel. The bedvolume is 10 ml with a void volume of 3.3 ml, and the total columnvolume is 30 ml. The gel is preconditioned by passing 20 mlphosphate-buffered saline (PBS, 10 ml sodium phosphate buffer, pH 7.4,containing 0.15 M sodium chloride) through the column. A volume of 0.5ml of the LDL stock solution is then applied to the column. After theLDL solution has run into the gel, 2.5 ml PBS is applied. The first 3 mlof eluate are discharged. The column is then eluted with 1 ml PBS, and 1ml EDTA-free LDL solution is collected in a 1.5-ml Eppendorf vial. Thevial is immediately made oxygen-free by nitrogen gassing and transferredto a refrigerator. An aliquot is removed to determine again theconcentration by the CHOD-PAP method. The LDL solution can be ratherunstable at this stage, depending on the donor, and therefore the timeelapsed between desalting and the final oxidation experiment should notexceed 60 mm.

Thiobarbituric Acid-Reactive Substances as Index of Low-DensityLipoprotein Oxidation

The preferred assay in LDL oxidation studies, both in presence andabsence of cells, is the determination of thiobarbituric acid(TBA)-reactive substances (TBARS) by one of the TBA assays developed forlipid peroxidation studies. The basal value of TBARS in freshly preparedLDL samples is usually low (0.5 to 3 nm/mg LDL protein) or undetectable.In LDL oxidized for about 24 hr with cells or CU²⁺ ions, the TBARS arein the range of 30 to 100 nmol/mg protein. In copper-stimulatedoxidation, formation of TBARS shows a lag phase of about 40-150 mindepending on the LDL, temperature, medium, and Cu2+ concentration;during this lag phase TBARS do not increase. Thereafter, TBARS rapidlyincrease for about 1-2 hr to a plateau value. On prolonged incubationTBARS remain more or less constant or increase slightly. The reportedtime course studies for TBARS in cell-mediated oxidation suggest thatoxidation proceeds similarly to Cu²⁺ oxidation, with a lag phasefollowed by a rapid increase to a plateau level. In this context, itshould be noted that most researchers only determine TBARS as an endpoint after about 24 hr incubation, when LDL has reached a final stageof oxidation.

Assays Used for Measurement of TBARS

Specifically, 100 ul of an LDL preparation (50 ug LDL cholesterin or 150ug protein) is added to 1 ml of 20% trichloroacetic acid (TCA).Following precipitation, 1 ml of 1% thiobarbituric acid (TBA) is added,and the mixture is heated 45 min at 95°, cooled on ice, and centrifuged(20 min at 1000 g). TBARS are then determined by measuring theabsorbance at 532 nm or the emission fluorescence at 553 nm (excitation515 nm). Calibration is done with a malonaldehyde standard prepared fromtetramethoxypropane.

The second assay is typically as follows: LDL (25 ug protein) is mixedwith 1.5 ml of 20% TCA and 1.5 ml of 0.67% TBA. After heating at 100°for 30 mm, TBARS are determined fluorimetrically at an emissionwavelength of 553 nm with excitation at 515 nm. The sensitivity wasreported to be 0.1 nmol TBARS/assay. This is equivalent to 4 nmolTBARS/mg protein. Haberland a al. determined the malonaldehyde-LDLadduct using a TBA assay. The malondialdehyde (MDA-treated LDL wasprecipitated with heparin-manganese, the supernatant was dischargedafter centrifugation, and the precipitate was washed withheparin-manganese prior to the TBA test.

Minimally modified LDL (MM-LDL) is prepared by dialyzing native LDLagainst 9 uM FeSO₄ in PBS for 72 h at 4° C. The electrophoretic mobilityincreased 1.1 to 1.2 versus native LDL. Mildly oxidized LDL was alsoobtained by (UV+copper/EDTA)-mediated oxidation under mild conditions:LDL solution (2 mg of apo/B/ml containing 2 umol/liter CuSO4) wasirradiated for 2 h. as a thin film (5 mm) in an open beaker placed 10 cmunder the UV-C source (HNS 30W OFR Osram UV-C tube, 1_(max) 254 nm 0.5milliwatt/cm² determined using a Scientech thermopile Model 360001),under the standard conditions. At the end of the irradiation, aliquotswere taken up for analyses and oxidized LDL (200 ug of apoB/ml understandard conditions or at the indicated concentration) were immediatelyincorporated in the culture medium.

Acetylation of LDL is performed with excess acetic anhydride. Endotoxincontamination in oxyLDL is measured with the coagulation Limulasamebocyte lysate assay using a commercially available kit (E-TOXATE,Sigma Chemical Co.).

Induction of Apoptosis by oxyLDL

Incubation of HUVEC with oxLDL for 18 hours induced DNA fragmentation ina concentration-dependent manner with maximal effects at 10 ug/mL. Incontrast, native LDL did not induce apoptosis in the concentration rangetested. The induction of apoptosis by oxyLDL is confirmed bydemonstrating DNA fragmentation through agarose gel electrophoresis. LDHrelease did not increase ³10 ug/mL oxLDL (105±11% compared with controlcells) excluding the induction of necrosis

Detection of Fas and FasL Expression on Endothelial Cells.

90% confluent HAECs and HUVECs were incubated with oxyLDL (150 ugprotein/ml) or L-a-palmitoyl lysophosphatidyleholine (LPC, 45 uM, SigmaChemical Co.) at 37° C., 5% CO2 for 13 h, and detached from the cultureplate with 0.5% EDTA. To determine FasL expression, endothelial cellsare incubated with an anti-FasL antibody (C-20, Santa CruzBiotechnology, Santa Cruz, Calif.) or with rabbit IgG followed by aFITC-conjugated antibody against rabbit Ig (Biosource, Camarillo,Calif.). To determine Fas expression, endothelial cells were incubatedwith an FITC-conjugated anti-Fas monoclonal antibody (clone UBZ.Immunotech. Wemtbrook. Me.) or an FITC.conjugated mouse IgG.Immunofluorescence staining was analyzed by FACS (fluorescence-activatedcell sorter) (Becton Dickinson. Mountain View, Calif.).

Detection of DNA Fragmentation by Agarose Gel Electrophoresis.

HUVECs (10⁶) wcre incubated in the presence or absence of native LDL(300 ug protein/ml). oxyLDL (300 ug protein/ml). LPS (100 endotoxinU/ml), or a neutralizing anti-FasL antibody (i0 ug/ml, 4H9, MBL, Nagoya.Japan) for 36 h. Attached cells and floating cells were combined andlysed in 0.33 ml of lysis buffer (10 mM Tris-HCi. pH 8.0, 1 mM EDTA,0.2% Triton X-100) followed by incubation with 0.1 mg/ml RNAase A for 1h at 37° C. and 0.2 mg/mi proteinase K for 3 h at 50° C.Ethanol-precipitated DNA was resuspended in TE buffer, fractionated on1.5% agarose gel in IX THE buffer, and stained with ethidium bromide.

Detection of DNA Fragmentation by TdT-Mediated dUTP Nick-End Labeling(TUNEL).

70% confluent HUVECs are incubated in the presence or absence of OxLDL(300 ug protein/ml), a neutralizing anti-FasL antibody (10 ug/mi. 4H9).or an agonistic anti-Fas antibody (0.5 ug/ml CH11, MBL) for 16 h at 37°C., 5% CO₂. Attached cells harvested by trypsinization and floatingcells are combined, fixed in 4% paraformaidehyde, permeabilized in 0.1%Triton X-100, 0.1% sodium citrate, and incubated with TUNEL solution(Boehringer Mannheim. Indianapolis. Ind.) in the absence or in thepresence of terminal deoxynucleotidyl transferase. After washing in PBS,fluorescence intensity was analyzed by FACS.

Cell Viability Assay.

HAECs or HUVECs are cultured in a 96-well plate at 80% confluency andincubated in the presence or absence of oxyLDL (300 ug protein/ml),LPC-C16:0 (55 uM). a neutralizing anti-FasL antibody (10 ug/ml. 4H9). oran agonistic anti-Fas antibody (0.5 ug/ml. CHI 1) for 18 h. Cellviability is measured by means of MTT(dimethyithiazol-diphenyltetrazolium bromide) assay and percentage ofcell death was calculated as 100×(1−viability of treated endothelialcells/viability of untreated endothelial cells).

Cell Viability Assay and Reagents—Human umbilical vein endothelial cells(HUVECs) are isolated and cultured in endotheial growth medium (SCM;Clonetics, San Diego, Calif.). HUVECs cultured in a 96-well plate at 80%confluency are incubated with oyxLDL or LPC at indicated doses for 16 h.Cell viability is measured by means of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay

EXAMPLE 50 Preparation of Vesicles Expressing Recombinant Membrane BoundSuperantigens Using the Yeast sec6 Mutant

The superantigen cDNA, or oxyLDL receptor, apoprotein verotoxin or otherpolypeptide given herein corresponding to the cDNA for proteinexpression in yeast is used. The length of the 5′-untranslated region isminimized. Expression of a cDNA in the sec6-4 yeast mutant is bestcontrolled and may be maximized with an inducible promoter. The GALlpromoter is preferred. The pYES2 expression vector (InVitrogen, SanDiego, Calif.) contains the GALI promoter followed by a multiple cloningsite. Other commonly used inducible promoters include themetallothionein CUPI promoter, which is tightly controlled by copper;promoters activated in response to heat shock, which are of particularinterest for expression in the temperature-sensitive sec6-4 mutant andthe PH05 promoter, which is derepressed at low phosphate concentrations.Introduction of the plasmid into yeast cells is accomplished either byelectroporation or LiCi-mediated transformation. Isolation oftransformants requires selection yeast that are ura3 auxotrophs are ableto grow on media lacking uracil when they contain the pYES2 expressionvector that contains the wild-type URA3 gene. Other selectable markersinclude enzymes in the adenine, histidine, leucine, lysine, andtryptophan biosynthetic pathways. The superantigen cDNAs are cloned intothe pYES2 expression vector and selected for transformants on plateswith synthetic complete (SC) medium lacking uracil but containing 2%raffmose as the carbon source (SC-Ura raff medium). Single colonies areisolated and grown overnight to saturation in 2 ml of SC-Ura raff mediumat 25° with constant shaking in 2% raffinose instead of glucose. In asubsequent step the yeast are switched to medium containing galactose asthe carbon source as the GALl promoter initiates gene expression onlywhen galactose is the predominant carbon source. The 2-ml starterculture in SC-Ura raff medium is added to a 1-liter culture of the samegrowth medium and incubated at 25° with constant shaking. When thesecultures reach an OD600 (optical density at a wavelength of 600 nm) ofabout 1.0 (usually about 12 hr), the cultures are centrifuged at 4000 gat 4° for 5 min, resuspended in 4 liters of SC-Ura gal induction medium(containing 2% galactose instead of 2% raffinose as the carbon source),and shifted to 37° for 2-3 hr to induce protein expression in the sec6vesicles.

Following growth at 37° the cells are collected by centrifugation at4000 g at 40 for 5 mm and washed once in ice-cold water. Pellets areresuspended in an absolute minimum volume of water and quick frozen inliquid nitrogen. Cultures may then be stored indefinitely at −70°.Thawed cultures are resuspended to a final concentration of 50OD618˜units/ml (e.g., a 1-liter culture at OD600=1.0 is resuspended in20 ml) in 10 mM dithiothreitol (DTF) and 100 mM Tris-CI, pH 9.4. Theresuspended culture is shaken gently at room temperature for 10 min.This step increases the efficiency of spheroplast lysis at a later stepby reducing disulfide bonds in the yeast cell wall. We then collect thecells by centrifugation at 4000 g at 4° for 5 mm and resuspend them inspheroplast buffer to a final concentration of 50 OD600 units/ml.Spheroplast buffer consists of 1.4 M sorbitol, 50 mM K2HPO4, pH 7.5, 10mM NaN3, and 40 mM 2-mercaptoethanol. Spheroplasts are generated bydigesting the cell wall with lyticase (or zymolyase) for 45 min at 37°,The amount of bacterially expressed, recombinant lyticase needed to formspheroplasts is determined empirically; after 45 min the OD₆₀₀ of a10-ul aliquot of the yeast suspension diluted into 1 ml of 0.1% sodiumdodecyl sulfate (SDS) should be ˜20% of the OD₆₀₀ of the initialdilution measured at 0 min. The spheroplasts are then harvested at 3000g for 5 min at 4°, and the cells are resuspended gently with a pipetteor Teflon rod in spheroplast buffer containing 10 mM MnC12 to a finalconcentration of 50 OD₆₀₀ units/ml. Concanavalin A (Sigma, St. Louis,Mo.) is then added to a final concentration of 0.78 to 1.25 mg/ml andincubated with rotation or gentle shaking at 4° for 15-30 mm. Aconcanavalin A stock solution (25 mg/ml) is prepared in spheroplastbuffer containing 1 mM MnCl2 and 1 mM CaCl₂ and is frozen in 1-mialiquots. Lectin-coated spheroplasts are harvested at 3000 g for 5 mm at4° and then resuspended in lysis buffer to a final concentration of60-70 0D₆₀₀ units/mi. The suspension is homogenized using the loosepestle of a Dounce homogenizer and 30-40 strokes of the pestle at 40 (oron ice). Lysis bufferconsists of 0.8 M sorbitol, 10 mM triethanolamine(TEA), and 1 mM EDTA. The pH is adjusted to 7.2 with acetic acid or TEA.Unlysed cells, cell debris, mitochondria, and nuclei are pelleted at20,000 g for 10 mm at 4° The supernatant is removed with a pipette andcentrifuged at 144,000 g for 1 hr at 40 to pellet the secretoryvesicles. The supernatant is decanted carefully and the pellet isresuspended in either lysis buffer or another buffer containing osmoticsupport.

Additional Documents Incorporated by Reference

This application incorporates by reference the following patents andcurrently pending patent applications that disclose inventions of thepresent inventor alone or with co-inventors.

-   1. Patent application WO91/US342, “Tumor Killing Effects of    Enterotoxins and Related Compounds” filed 17 Jan. 1991, and    published as WO 91/10680 on 25 Jul. 1991.-   2. U.S. Ser. No. 07/891,718 “Tumor Killing Effects of Enterotoxins    and Related Compounds,” filed 1 Jun. 1992.-   3. U.S. Pat. No. 5,728,388, “Method of Cancer Treatment,” issued    Mar. 17, 1998.-   4. U.S. Ser. No. 08/491,746, “Method of Cancer Treatment,” filed 19    Jun. 1995.-   5. U.S. Ser. No. 08/898,903 “Method of Cancer Treatment,” filed 23    Jul. 1997.-   6. U.S. Ser. No. 08/896,933 “Tumor Killing Effects of Enterotoxins    and Related Compounds,” filed 18 Jul. 1997.-   7. U.S. Ser. No. 60/085,506, “Compositions and Methods for Treatment    of Cancer,” filed 5 May 1998.-   8. U.S. Ser. No. 60/094,952 “Compositions and Methods for Treatment    of Cancer” filed 31 Jul. 1998.-   9. U.S. Ser. No. 60/033,172 “Superantigen-Based Methods and    Compositions for Treatment of Cancer,” filed 17 Dec. 1996.-   10. U.S. Ser. No. 60/044,074 “Superantigne-Based Methods and    Compositions for Treatment of Cancer,” filed 17 Apr. 1997.-   11. U.S. Ser. No. 09/061,334 “Tumor Cells with Increased    Immunogenicity and Uses Thereof,” filed 17 Apr. 1998.

Moreover, all references cited herein are incorporated by reference,whether specifically incorporated or not.

Having now fully described this invention, it will be appreciated bythose skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations, and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation.

While this invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications. This application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth as follows in the scope of theappended claims.

1. A mammalian cell comprising an exogenous nucleic acid encoding asuperantigen which is expressed in said cell which cell also produces orexpresses all α-anomers of monoglycosylceramide or diglycosylceramide,wherein expression of said superantigen and said mono- ordi-glycosylceramide is capable of eliciting an effective anti-tumorimmune response in a mammal into which said cell is introduced.
 2. Thecell of claim 1 which is selected from a group consisting of (a) a tumorcell (b) an accessory cell (c) a tumor cell/accessory cell hybrid
 3. Thecell of claim 1 wherein said mammal bears a tumor, against which theantitumor response is directed, said tumor being selected from the groupconsisting of a carcinoma, a melanoma, a sarcoma, a neuroblastoma, anastrocytoma, a lymphoma and a leukemia.
 4. The cell of claim 1 whereinthe superantigen is selected from the group consisting of aStaphylococcal enterotoxin and a Streptococcal pyrogenic exotoxin.
 5. Amethod of treating a tumor or neoplastic disease in a subject,comprising administering to said subject an effective amount of thecells of any of claims 1-4, wherein said nucleic acid is introduced invivo into a cell that produces or expresses said mono- ordiglycosylceramide.
 6. A method of treating a tumor or neoplasticdisease in a subject comprising administering an effective amount of thecells of any of claims 1-4 wherein said superantigen exogenous nucleicacids is introduced ex vivo or in vitro into a cell which produces orexpresses said mono- or di-glycosylceramide.
 7. A composition useful fortreating a tumor or neoplastic disease in a subject comprising aconjugate or complex of (a) a superantigen; and (b) a glycosylceramide.8. The composition of claim 7 wherein the glycosylceramide is selectedfrom a group consisting of (a) an α-1-4-galabiosylceramide, (b) anα-1-4-globotriosylceramide, (c) an α-1-4-globoteterasylceramide or (d) aglycosylphosphatidylinositol-anchored α-1-4-galabiosylceramide, (e) aglycosylphosphatidylinositol-anchored α-1-4-globotriosylceramide, and(f) a glycosylphosphatidylinositol-anchoredα-1-4-globoteterasylceramide.
 9. The composition of claim 7, wherein theglycosylceramide comprises a phytosphingosine chain having unsubstitutedhydroxyl groups at its C3- and C4 position.
 10. The composition of claim7, wherein the length of the ceramide fatty acyl chain is from about 12to about 24 carbons.
 11. The composition of claim 7, wherein thesphingosine portion of the ceramide has a chain length of about 10 toabout 13 carbons.
 12. The composition of claim 7, wherein the conjugateor complex further comprises CD1 receptors, MHC class I molecules, MHCclass II molecules or superantigen receptors.
 13. The composition ofclaim 7-12 wherein the superantigen-glycosylceramide is in or on avesicle, exosome, liposome, phage display, prokaryotic cell surface oreukaryotic cell surface.
 14. The composition of claims 7-12 wherein saidcomplex or conjugate is obtained by shedding from cells expressing saidcomplexes or conjugates.
 15. The composition of claim 7-12 wherein thesuperantigen-glycosylceramide conjugate, or, if present, saidsuperantigen-GPI-glycosylceramide conjugate, is chemically linked by acrosslinking agent
 16. The compositions of claims 13 wherein thesuperantigen-glycosylceramide is loaded onto a prokaryotic or eukaryoticcell surface.
 17. A method of treating a tumor or neoplastic disease ina subject comprising administering to the subject an effective amount ofcells loaded with the compositions of any of claims 7-12, so that thecells present the composition to the immune system, thereby inducing ananti-tumor immune response.
 18. A method of treating a tumor orneoplastic disease in a subject comprising administering to the subjectan effective amount of compositions of claim 13, thereby inducing ananti-tumor immune response.
 19. A method of treating a tumor orneoplastic disease in a subject comprising administering to the subjectan effective amount of the composition of claim 15, thereby inducing ananti-tumor immune response.
 20. The method of claim 17 wherein thecomposition is a superantigen-glycosylceramide-CD1 conjugate or complexwherein the glycosylceramide is selected from a group consisting of (a)an α1-4-galabiosylceramide, (b) an α-1-4-globotriosylceramide, (c) anα-1-4-globoteterasylceramide or (d) aglycosylphosphatidylinositol-anchored α-1-4-galabiosylceramide, (e) aglycosylphosphatidylinositol-anchored α-1-4-globotriosylceramide, and(f) a glycosylphosphatidylinositol-anchoredα-1-4-globoteterasylceramide.
 21. The method of claim 18 wherein thecomposition is a superantigen-glycosylceramide-CD1 conjugate or complexwherein the glycosylceramide is selected from a group consisting of (a)an α1-4-galabiosylceramide, (b) an α-1-4-globotriosylceramide, (c) anα-1-4-globoteterasylceramide or (d) aglycosylphosphatidylinositol-anchored α-1-4-galabiosylceramide, (e) aglycosylphosphatidylinositol-anchored α-1-4-globotriosylceramide, and(f) a glycosylphosphatidylinositol-anchoredα-1-4-globoteterasylceramide.
 22. The method of claim 17 wherein thecells are selected from the group consisting of: (a) dendritic cells;(b) macrophages or monocytes; (c) fibroblasts; (d) keratinocytes; (e)stromal cells; (f) antigen presenting cell; (g) tumor cells; (h)lymphocytes; and (i) a combination of any two or more of (a)-(h). 23.The method of claim 18 wherein the cells are selected from the groupconsisting of: (a) dendritic cells; (b) macrophages or monocytes; (c)fibroblasts; (d) keratinocytes; (e) stromal cells; (f) antigenpresenting cell; (g) tumor cells; (h) lymphocytes; and (i) a combinationof any two or more of (a)-(h).
 24. The method of claim 19 wherein thecells are selected from the group consisting of: (a) dendritic cells;(b) macrophages or monocytes; (c) fibroblasts; (d) keratinocytes; (e)stromal cells; (f) antigen presenting cell; (g) tumor cells; (h)lymphocytes; and (i) a combination of any two or more of (a)-(h). 25.The method of claim 20 wherein the cells are selected from the groupconsisting of: (a) dendritic cells; (b) macrophages or monocytes; (c)fibroblasts; (d) keratinocytes; (e) stromal cells; (f) antigenpresenting cell; (g) tumor cells; (h) lymphocytes; and (i) a combinationof any two or more of (a)-(h).
 26. The compositions of claim 7-12,wherein the superantigen is selected from the group consisting of aStaphylococcal enterotoxin and a Streptococcal pyrogenic exotoxin.
 27. Amethod of treating a tumor or neoplastic disease in a subject comprisingadministering to said subject an effective amount of the composition ofany of claims.
 28. A method of preparing a population ofimmunotherapeutically active T or NKT cells useful to treat a tumor orneoplastic disease in a subject, comprising: (a) providing to (i) asubject in vivo or (ii) a population of T and/or NKT cells ex vivo or invitro  the cells of any of claims 1-4 or a composition of any of claims7-12 to prime or stimulate the production of a population oftumor-specific T cells and/or NKT cells, (b) obtaining said primed orstimulated T or NKT cells; (c) optionally, further contacting saidprimed or stimulated T or NKT cells with any of said cells orcompositions ex vivo to expand and further stimulate said T or NKTcells. thereby preparing said of immunotherapeutically active cells. 29.A method of treating a tumor or neoplastic disease in a subject,comprising administering an effective amount of T and/or NKT cellsprepared in accordance with claim 24 to said subject to treat said tumoror neoplastic disease.
 30. A composition useful for treating a tumor orneoplastic disease in a subject comprising naked DNA encoding asuperantigen conjugated to a protein which induces apoptosis of tumorcells in said subject.
 31. The composition of claim 25 wherein theprotein is selected from a group consisting of (a) Fas; (b) Perforin;(c) Granzyme B; (d) Tumor Necrosis Factor α or □; (e) Verotoxin; and (f)a Verotoxin A chain, B chain or hybrid AB chain.
 32. A compositionuseful for treating a tumor or neoplastic disease in a subjectcomprising naked DNA encoding a superantigen conjugated to a verotoxin.33. A composition useful for treating a tumor or neoplastic disease in asubject comprising naked DNA encoding a superantigen conjugated to aprotein or peptide that has at least about 30% sequence identity to theGal(α1-4)Gal-binding region of a verotoxin.
 34. A composition useful fortreating a tumor or neoplastic disease in a subject comprising naked DNAencoding a superantigen conjugated to a protein or peptide that has atleast about 45% sequence identity to the Gal(α1-4)Gal-binding region ofa verotoxin.
 35. The composition of claim 33 or 34 wherein the peptideor protein has the amino acid sequence of all or part of anyGal(α1-4)Gal-binding portion of: (a) the 63 kDa extracellular peptide ofthe interferon α receptor; or (b) the N terminal extracellular domain ofCD19.
 36. An apoptotic cell preparation or lysate useful for treating atumor or neoplastic disease in a subject, comprising a cell populationthat has been (a) transfected with naked DNA encoding a superantigen;and (b) treated to undergo apoptosis or lysis.
 37. A cell which hasingested or been transfected with the apoptotic preparation or lysate ofclaim 36, thereby rendering the cell effective in presenting materialexpressed from transfecting nucleic acid or material ingested to theimmune system of a mammal to elicit an anti-tumor immune response.
 38. Amethod for treating a tumor or neoplastic disease in a subject,comprising (a) providing to a population of cells selected from thegroup consisting of: (i) tumor cells; (ii) accessory cells; (iii) tumorcell/accessory cell hybrids; (iv) cells with an inherent or acquired-galactosidase deficiency; and (v) a combination of any two or more of(i)-(iv), said apoptotic cell preparation or said lysate of claim 36, toproduce an immunostimulatory cell population; (b) administering to saidsubjected an amount of said immunostimulatory cell population effectiveto treat said tumor or neoplastic disease.
 39. The method of claim 38wherein said accessory cells are dendritic cells and said hybrid cells(iii) are dendritic cell/tumor cell hybrids.
 40. The method of claim 38wherein the providing step (a) is in vivo.
 41. The method of claim 38wherein the providing step (a) is in vitro.
 42. A method of treating atumor or neoplastic disease in a subject comprising administering tosaid subject an effective amount of cells according to claim 36, whereinsaid ingested lysate, transfecting nucleic acid or other apoptoticmatter is presented to the immune system to elicit a tumoricidalresponse.
 43. A composition useful for treating a tumor or neoplasticdisease in a subject comprising a lipoprotein which is capable ofbinding to receptors in tumor microvasculature and eliciting apoptosisof tumor endothelial cells and eliciting an effective anti-tumorresponse in a mammal into which said lipoprotein is introduced.
 44. Thecomposition of claim 43 wherein the lipoprotein is selected from thegroup consisting of: (a) low density lipoproteins (a) chylomicrons (b)very low density lipoproteins (c) apolipoproteins (d) oxidized lowdensity lipoproteins (e) oxidized low density lipoprotein byproducts (f)oxidized low density lipoproteins mimics (g) low density lipoproteincomplexed with compounds which enhance or promote the uptake by cellsexpressing LDL or oxidized LDL receptors.
 45. The composition of claim44 wherein the compounds which enhance or promote the uptake by cellsexpressing LDL or oxidized LDL receptors are selected from a groupconsisting of: (a) fibronectin (b) collagen (c) heparan
 46. Acomposition useful for treating a tumor or neoplastic disease in asubject comprising a conjugate or complex of: (a) a superantigen; and(b) a lipoprotein
 47. The composition of claim 46 wherein thelipoprotein is selected from the group consisting of: (a) low densitylipoproteins (a) chylomicrons (b) very low density lipoproteins (c)apolipoproteins (d) oxidized low density lipoproteins (e) oxidized lowdensity lipoprotein byproducts (f) oxidized low density lipoproteinsmimics (g) low density lipoprotein complexed with compounds whichenhance or promote the uptake by cells expressing LDL or oxidized LDLreceptors.
 48. the composition of claim 47 wherein the compounds whichenhance or promote the uptake by cells expressing LDL or oxidized LDLreceptors are selected from a group consisting of: (a) fibronectin (b)collagen (c) heparan
 49. The compositions of claims 46-48 wherein thesuperantigen-lipoprotein conjugate is in or on a vesicle, exosome,liposome, phage display, prokaryotic cell surface or eukaryotic cellsurface.
 50. The compositions of claim 49 which are are derived from agroup consisting of: (a) a mammalian cell transfected with superantigengenes (b) a sickle cell or sickle cell precursor transfected withsuperantigen genes (c) a yeast cell or mutant transfected withsuperantigen genes (d) a Staphylococcus carnosus transfected withsuperantigen genes. (e) a Sphingomonas paucimobilis transfected withsuperantigen genes 51 The compositions of claims 46-50 wherein thesuperantigen is selected from the group consisting of a Staphylococcalenterotoxin and a Streptococcal pyrogenic exotoxin.
 52. The method oftreating a tumor or neoplastic disease in a subject comprisingadministering to the subject an effective amount of the compositions ofclaims 46-50 so that the composition localizes in tumor microvasuclatureand is presented to the immune system, thereby inducing an anti-tumorresponse
 53. A mammalian cell useful for treating a tumor or neoplasticdisease in a subject comprising a nucleic acid encoding a receptor forLDL or oxidized LDL which renders the said cell capable of binding LDLor oxyLDL and undergoing apoptosis and eliciting an effective anti-tumorresponse.
 54. The mammalian cell of claim 53 comprising a secondexogenous nucleic acid encoding a superantigen such that the expressionof said superantigen and products of the first nucleic acid alone or incombination are capable of eliciting an effective anti-tumor immuneresponse.
 55. The cell of claim 53 is selected from a group consistingof: (a) tumor cells (b) endothelial cells (c) stromal cells
 56. The cellof claim 53 wherein the LDL or oxidized LDL receptor is selected for thegroup consisting of: (a) scavenger receptors expresssed on endothelialcells and macrophages (b) LOX-1 receptor (c) oxidized low densitylipoprotein receptor (d) CD36 receptor (e) Acetyl low densitylipoprotein receptor (f) low density lipoprotein receptor (g) lowdensity lipoprotein receptor-related protein (LRP)
 57. The cell of claim55 wherein the superantigen is selected from the group consisting of aStaphylococcal enterotoxin and a Streptococcal pyrogenic exotoxin.
 58. Amethod of treating a tumor or neoplastic disease in a subject comprisingadministering an effective amount of exogenous LDL receptor, oxyLDLreceptor nucleic acid or superantigen nucleic acid wherein they areintroduced into the cell in vivo.
 59. A composition useful for treatinga tumor or neoplastic disease in a subject comprising a conjugate orcomplex of: (a) a superantigen; and (b) a LDL or oxidized low densitylipoprotein receptor
 60. The composition of claim 59 wherein the LDL oroxidized LDL receptor is selected for the group consisting of: (a)scavenger receptors expresssed on endothelial cells and macrophages (b)LOX-1 receptor (c) oxidized low density lipoprotein receptor (d) CD36receptor (e) Acetyl low density lipoprotein receptor (f) low densitylipoprotein receptor (g) low density lipoprotein receptor-relatedprotein (LRP) (h) apolipoprotein receptors
 61. The compositions ofclaims 58-59 wherein the LDL and oxidized LDL receptors are in the formof naked DNA.
 62. The compositions of claims 59-60 wherein the LDLreceptor or superantigen or LDL receptor is a polypeptide or nucleicacid in or on a vesicle, exosome, liposome, phage display, plasmid,expression vector, prokaryotic cell surface or eukaryotic cell surface.63. The vesicles, exosomes prokaryotic and eukaryotic cell surfaces ofclaim 61 which are derived from a group consisting of: (a) a mammaliancell transfected with superantigen genes (b) a sickle cell or sicklecell precursor transfected with superantigen genes (c) a yeast cell ormutant yeast cell transfected with superantigen genes (d) aStaphylococcus carnosus transfected with superantigen genes. (e) aSphingomonas paucimobilis transfected with superantigen genes
 64. Thecompositions of claims 58-63 wherein the superantigen is selected fromthe group comprising a Staphylococcal enterotoxin and Streptococcalpyrogenic exotoxin
 65. A mammalian cell comprising an exogenous nucleicacid encoding a superantigen which is expressed in said cell which cellalso produces or expresses low density lipoproteins, wherein expressionof said superantigen and said native LDL or oxidized LDL or biologicallyactive LDL mimics and byproducts is capable of eliciting an effectiveanti-tumor immune response in a mammal into which said cell isintroduced.
 66. The cell of claim 65 which is selected from a groupconsisting of (a) a tumor cell (b) a endothelial cell (b) a sickled cellor sickled cell precursor
 67. The cell of claim 65 wherein the the lowdensity lipoproteins are selected from the group consisting of: (a)native low density lipoprotein (a) oxidized low density lipoprotein (b)low density lipoprotein mimics (c) low density lipoprotein byproducts68. The cell of claim 65 wherein said mammal bears a tumor, againstwhich the antitumor response is directed, said tumor being selected fromthe group consisting of a carcinoma, a melanoma, a sarcoma, aneuroblastoma, an astrocytoma, a lymphoma and a leukemia.
 69. A methodof treating a tumor or neoplastic disease in a subject, comprisingadministering to said subject an effective amount of the cells of any ofclaims.
 70. A method of treating a tumor or neoplastic disease in asubject comprising administering an effective amount of the cells of anyof claims 65-67 wherein said superantigen exogenous nucleic acids isintroduced ex vivo or in vitro into a cell which produces or expresseslow density lipoproteins
 71. A method of treating a tumor or neoplasticdisease in a subject comprising administering an effective amount of thecells of any of claims 65-67 wherein said superantigen exogenous nucleicacids and apolipoprotein nucleic acid is introduced ex vivo or in vitrointo a cell which thereby expresses superantigen and apolipoprotein. 72.The method of claim 70-71 wherin the low density lipoproteins areselected from a group consisting of: (a) native low density lipoprotein(a) oxidized low density lipoprotein (b) low density lipoprotein mimics(c) low density lipoprotein byproducts
 73. A mammalian cell comprisingexpressing a α-monogalactosylceramide or α-digalactosylceramide oroxidized low density lipoprotein individually or in any combinationwhich is (are) capable of binding to tumor microvasculature, inducingapoptosis and eliciting an effective anti-tumor immune response in amammal into which said cell is introduced.
 74. The cell of claim 73which also expresses a superantigen
 75. The cell of claims 73-74 whichis selected from a group consisting of: (a) a tumor cell (b) a sickledcell or sickled cell precursor
 76. A mammalian tumor cell/accessory cellhybrid cell comprising an exogenous nucleic acid encoding a superantigenwhich is expressed in said cell such that expression of saidsuperantigen renders the said cell capable of elicting an effectiveanti-tumor immune response in a mammal into which said cell isintroduced.
 77. The cell of claim 76 wherein said hybrid cell is adendritic cell/tumor cell hybrid.
 78. The cells of claims 73-77 whereinsaid mammal bears a tumor, against which the antitumor response isdirected, selected from the group consisting of a carcinoma, a melanoma,a sarcoma, a neuroblastoma, a lymphoma and a leukemia.
 79. The cell ofclaim 73-77 wherein the superantigen is selected from the groupconsisting of a Staphylococcal enterotoxin and a Streptococcal pyrogenicexotoxin.
 80. A method of treating a tumor or neoplastic disease in asubject comprising administering an effective amount of nucleic acids invivo to the tumor/accessory tumor/accessory cell hybrid cells of claims76-77.
 81. A method of treating a tumor or neoplastic disease in asubject comprising administering an effective amount of the cells ofclaim 76-77 wherein said exogenous nucleic acids are introduced into thecell ex vivo or in vitro.